Ion channel

ABSTRACT

The present invention relates to a novel 1,957 amino acid tetrodotoxin-insensitive voltage-gated sodium channel specifically located in mammalian sensory neurons. Nucleic acid sequences coding for the novel sodium channel, vectors, host cells and methods of identifying modulators of the novel sodium channel for use in treatment of pain are also provided.

Voltage-gated sodium channels are transmembrane proteins which causesodium permeability to increase. Depolarization of the plasma membranecauses sodium channels to open allowing sodium ions to enter along theelectrochemical gradient creating an action potential.

Voltage-gated sodium channels are expressed by all electricallyexcitable cells, where they play an essential role in action potentialpropagation. They comprise a major subunit of about 2000 amino acidswhich is divided into four domains (D1-D4), each of which contains 6membrane-spanning regions (S1-S6). The alpha-subunit is usuallyassociated with 2 smaller subunits (beta-1 and beta-2) that influencethe gating kinetics of the channel. These channels show remarkable ionselectivity, with little permeability to other monovalent or divalentcations. Patch-clamp studies have shown that depolarisation leads toactivation with a typical conductance of about 20 pS, reflecting ionmovement at the rate of 10⁷ ions/second/channel. The channel inactivateswithin milliseconds (Caterall, W. A., Physiol. Rev. 72, S4-S47 (1992);Omri et al, J. Membrane Biol 115, 13-29; Hille, B, Ionic Channels inExcitable Membranes, Sinauer, Sunderland, Mass. (1991)).

Sodium channels have been pharmacologically characterised using toxinswhich bind to distinct sites on sodium channels. The heterocyclicguanidine-based channel blockers tetrodotoxin (TTX) and saxitoxin (STX)bind to a site in the S5-S6 loop, whilst μ-conotoxin binds to anadjacent overlapping region. A number of toxins from sea anemones orscorpions binding at other sites alter the voltage-dependence ofactivation or inactivation.

Voltage-gated sodium channels that are blocked by nanomolarconcentrations of tetrodotoxin are known as tetrodotoxin sensitivesodium channels (Hille (1991) “Ionic Channels in Excitable Membranes”,Sinauer Sunderland, Mass. (1991)) whilst sodium channels that areblocked by concentrations greater than 1 micromolar are known astetrodotoxin-insensitive (TTXi) sodium channels (Pearce and DuchenNeuroscience 63, 1041-1056 (1994)).

Dorsal root ganglion (DRG) neurons express at least three types ofsodium channels which differ in kinetics and sensitivity to TTX. Neuronswith small-diameter cell bodies and unmyelinated axons (C-fibers)include most of the nociceptor (damage-sensing) population and express afast TTX-sensitive current and a slower TTX-insensitive current. Of thefive cloned sodium channel α-subunit transcripts known to be present indorsal root ganglia, none exhibits the properties of the TTX-insensitivechannel.

Sodium channel blockers are used clinically to provide pain relief.Three classes of sodium channel blockers in common clinical use are:local anesthetics such as lidocaine, some anticonvulsants such asphenytoin and carbamazepine, and some antiarrhythmics such asmexiletine. Each of these is known to suppress ectopic peripheralnervous system discharge in experimental preparations and to providerelief in a broad range of clinical neuropathic conditions.

Applicants have now found a novel voltage-gated sodium channel(hereinafter referred to as a sodium channel specifically located insensory neurons or also referred to as SNS sodium channel) that ispresent in sensory neurons (or neurones) but not present in glia,muscle, or the neurons of the sympathetic, parasympathetic, enteric orcentral nervous systems. Preferably the sodium channel of the inventionis found in the neurons of the dorsal root ganglia (DRG) or cranialganglia. More preferably the sodium channel of the invention is found inthe neurons of the dorsal root ganglia. Preferably the sodium channel isspecifically located in rat sensory neurons or human sensory neurons.

The sodium channel of the present invention is believed to play a rolein nociceptive transmission because some noxious input to the centralnervous system is known to be insensitive to TTX. Persistent activationof peripheral nociceptors has been found to result in changes inexcitability in the dorsal horn associated with the establishment ofchronic pain. Increased sodium channel activity has also been shown tounderlie neuroma-induced spontaneous action potential generation.Conversely, chronic pain may be successfully treated by surgical orpharmacological procedures which block peripheral nerve activation.Blockage of nociceptor input may therefore produce useful therapeuticeffects, even though central nervous system plasticity plays a pivotalrole in the establishment of chronic pain. Sensory neuron-specificvoltage-gated sodium channels, particularly sub-types associated with anociceptive modality such as the sodium channel of the invention, thusprovide targets for therapeutic intervention in a range of pain states.The electrophysiological and pharmacological properties of the expressedSNS sodium channel are similar to those described for the small diametersensory neuron tetrodotoxin-resistant sodium channels. As some noxiousinput into the spinal cord is resistant to tetrodotoxin, block ofexpression or function of such a C-fiber-restricted sodium channel mayhave a selective analgesic effect.

In another aspect the present invention provides an isolated proteincomprising a sodium channel specifically located in rat sensory neuronsas encoded by the insert deposited in NCIMB deposit number 40744, whichwas deposited at The National Collections of Industrial and MarineBacteria, 23 St Machar Drive, Aberdeen AB2 1RY, Scotland, United Kingdomon Jun. 27, 1995 in accordance with the Budapest Treaty.

The invention also provides nucleotide sequences coding for the SNSsodium channel. In a preferred embodiment, the nucleotide sequenceencodes a sodium channel specifically located in rat sensory neuronswhich is as set out in FIG. 1a or a complementary strand thereof.

The approximately 6.5 kilobase (kb) transcript expressed selectively inrat dorsal root ganglia that codes for the novel sodium channel of theinvention shows sequence similarities with known voltage-gated sodiumchannels. The cDNA codes for a 1,957 amino acid protein. In particular,the novel sodium channel of the invention shows 65% identity at theamino acid level with the rat cardiac tetrodotoxin-insensitive (TTXi)sodium channel. The aromatic residue that is involved in high-affinitybinding of TTX to the channel atrium of TTX-sensitive sodium channels isaltered to a hydrophilic serine in the predicted protein of the SNSsodium channel, whereas the residues implicated in sodium-selectivepermeability are conserved. The novel sodium channel specificallylocated in sensory neurons shows relative insensitivity to TTX (IC50>1micromolar) and thus exhibits properties different from other clonedsodium channel transcripts known to be present in dorsal root ganglia.

The invention also provides expression and cloning vectors comprising anucleotide sequence as hereinabove defined. In order to effecttransformation, DNA sequences containing the desired coding sequence andcontrol sequences in operable linkage (so that hosts transformed withthese sequences are capable of producing the encoded proteins) may beincluded in a vector, however, the relevant DNA may then also beintegrated into the host chromosome.

The invention also provides a screening assay for modulators of thesodium channel which is specifically located in sensory neurons whereinthe assay comprises adding a potential modulator to a cell expressingthe SNS sodium channel and detecting any change in activity of thesodium channel.

The present invention also provides a modulator which has activity inthe screening assay hereinabove defined. Modulators of the sodiumchannel as hereinabove defined are useful in modulating the sensation ofpain. Blockers of the sodium channel will block or prevent thetrasmission of impulses along sensory neurons and thereby be useful inthe treatment of acute, chronic or neuropathic pain.

The present invention thus relates to novel voltage-gated sodium channelproteins specific to sensory neurons, to nucleotide sequences capable ofencoding these sodium channel proteins, to vectors comprising anucleotide sequence coding for a sodium channel of the invention, tohost cells containing these vectors, to cells transformed with a nucleicacid sequence coding for the sodium channel, to screening assays usingthe sodium channel proteins and/or host cells, to complementary standsof the DNA sequence which is capable of encoding the sodium channelproteins and to antibodies specific for the sodium channel proteins.These and other aspects of the present invention are set forth in thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows the nucleic acid and amino acid sequences of the sodiumchannel specific to the rat DRG (SNS-B) (SEQ ID NO: 1 and SEQ ID NO: 2).

FIG. 1b shows the structure of the SNS-B voltage-gated sodium channel inpGEM-3Z.

FIG. 1c shows a schematised drawing of a known voltage-gated sodiumchannel.

FIG. 2 shows sequences of examples of PCR primers for isolation of humanclone probes. RLLRVFKLAKSWPTL-SEQ ID NO: 21; 5′ gcttgctgcgggtcttcaagc 3′SEQ ID NO: 22; LRALPLRALSRFEG-SEQ ID NO: 23; 5′atcgagacagagcccgcagcg3′SEQ ID NO: 24; 5′ acgggtgccgcaaggacggcgtctccgtgtggaacggcgagaag 3′ SEQ IDNO: 25; and 5′ ggctatccttcctcttccagctctcacccaggtatggagccaggt3′-SEQ IDNO: 26.

FIG. 3 shows a film of ³⁵S radio-labelled SNS-B voltage-gated sodiumchannel protein in a coupled transcription/translation system.

FIGS. 4a and 4 b show SNS-GST fusion protein constructs for antibodygeneration. TCCCGTACGCTGCAGCTCTTT-SEQ ID NO: 27; CCCGGGGAAGGCTAC-SEQ IDNO: 28; GTCGACACCAGAAAT-SEQ ID NO: 29;GGATCCTCTAGAGTCGACCTGCAGAAGGAA-SEQ ID NO: 30

In accordance with one aspect of the invention there is provided anisolated and/or purified nucleic acid sequence (or polynucleotide ornucleotide sequence) which comprises a nucleic acid sequence whichencodes the mammalian sodium channel specifically located in sensoryneurons or a complementary strand thereof. Preferably, the nucleic acidsequence encodes the sodium channel specifically located in mammaliandorsal root ganglia. More preferably, the nucleic acid sequence encodesthe rat or human sodium channel specifically located in dorsal rootganglia. The rat nucleic acid sequence preferably comprises the sequenceof the coding portion of the nucleic acid sequence shown in FIG. 1a (SEQID NO:1) or the coding portion of the cDNA deposited in NCIMB depositnumber 40744 which was deposited at the National Collections ofIndustrial and Marine Bacteria, 23 St. Machar Drive, Aberdeen AB21RY,Scotland, United Kingdom on Jun. 27, 1995 in accordance with theBudapest Treaty.

A nucleic acid sequence encoding a sodium channel of the presentinvention may be obtained from a cDNA library derived from mammaliansensory neurons, preferably dorsal root ganglia, trigeminal ganglia orother cranial ganglia, more preferably rat or human dorsal root ganglia.The nucleotide sequence described herein was isolated from a cDNAlibrary derived from rat dorsal root ganglia cells. The nucleic acidsequence coding for the SNS sodium channel has an open reading frame of5,871 nucleotides encoding a 1,957 amino acid protein. A nucleic acidsequence encoding a sodium channel of the present invention may also beobtained from a mammalian genomic library, preferably a human or ratgenomic library. The nucleic acid sequence may be isolated by thesubtraction hybridization method described in the examples, by screeningwith a probe derived from the rat sodium channel sequence, or by othermethodologies known in the art such as polymerase chain reaction (PCR)with appropriate primers derived from the rat sodium channel sequenceand/or relatively conserved regions of known voltage-gated sodiumchannels.

The nucleic acid sequences of the present invention may be in the formof RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the rat SNS sodium channel orvariant thereof may be identical to the coding sequences set forthherein or that of the deposited clone, or may be a different codingsequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same protein as thesequences set forth herein or the deposited cDNA.

The nucleic acid sequence which encodes the SNS sodium channel mayinclude: only the coding sequence for the full length protein or anyvariant thereof; the coding sequence for the full length protein or anyvariant thereof and additional coding sequence such as a leader orsecretory sequence or a proprotein sequence; the coding sequence for thefull length protein or any variant thereof (and optionally additionalcoding sequence) and non-coding sequences, such as introns or non-codingsequences 5′ and/or 3′ of the coding sequence for the full lengthprotein.

The present invention further relates to variants of the hereinabovedescribed nucleic acid sequences which encode fragments, analogs,derivatives or splice variants of the SNS sodium channel. The variant ofthe SNS sodium channel may be a naturally occurring allelic variant ofthe SNS sodium channel. As known in the art, an allelic variant is analternate form of a protein sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded protein. The presentinvention relates to splice variants of the SNS sodium channel thatoccur physiologically and which may play a role in changing theactivation threshold of the sodium channel.

Variants of the sequence coding for the rat SNS sodium channel have beenidentified and are listed below:

1) a 2573 base pair nucleic acid sequence shown in SEQ ID NO:3. Thissequence codes for a 521 amino acid protein that corresponds to aminoacids 1437-1957 of FIG. 1a (SEQ ID NO:1) and has the same sequence asbases 4512 through 6524 of FIG. 1a in the coding portion and 3′untranslated region.

2) a 7052 base pair nucleic acid sequence shown in SEQ ID NO: 5. SEQ IDNO:5 codes for a 2,132 amino acid protein that contains a 176 amino acidrepeat (amino acids 586-760 of SEQ ID NO:6) inserted after amino acid585 in FIG. 1a or SEQ ID NO:2.

A preferred sequence for the rat SNS sodium channel is shown in FIG. 1a(SEQ ID NO: 1). However, sequencing variations have been noted.Sequencing has provided

a 6,321 base pair nucleic acid sequence coding for a 1957 amino acidprotein that has the same base sequence as bases 1-6321 of FIG. 1a orSEQ ID NO:1 with the following changes: bases 1092 G to A, base 1096 Cto T, base 2986 G to T, base 3525 C to G and base 3556 G to C.

a 6,527 base pair nucleic acid sequence coding for a 1,957 amino acidprotein as shown in SEQ ID NO:7 that has the same base sequence as bases1-6524 of FIG. 1a (SEQ ID NO:1) with an additional 3 bases AAA, at the3′ end, and the following changes: base 299 C to G, base 1092 G to A,base 1096 C to T, base 1964 G to C, base 1965 C to G, base 2472 A to T,base 2986 G to T, base 3019 A to G, base 3158 C to T, base 3525 C to G,base 3556 G to C and base 5893 T to G. The sequence of SEQ ID NO: 7 isalso a preferred sequence coding for the rat SNS sodium channel.

a 6524 base pair nucleic acid sequence that has the same sequence asFIG. 1a (SEQ ID NO: 1) except for the following base changes: base 1092G to A (resulting in a change at amino acid 297 of SEQ ID NO: 2 from Valto Ile), base 1096 C to T (resulting in a change at amino acid 298 fromSer to Phe), base 1498 C to A (resulting in a change at amino acid 432from Ala to Glu), and base 2986 G to T (resulting in a change at aminoacid 928 form Ser to Ile).

Sequence variability has been identified in different isolates. One suchseqeuence has been identified that has the sequence of the thirdsequencing variation shown immediately above except for eight basedifferences, five of which resulted in an altered amino acid sequenceF16-S16, L393-P393, T470-1470, R278-H278, and I1,876-M1,876.

The present invention also relates to nucleic acid probes constructedfrom the nucleic acid sequences of the invention or portion thereof.Such probes could be utilized to screen a dorsal root ganglia cDNAlibrary to isolate a nucleic acid sequence encoding the sodium channelof the present invention. The nucleic acid probes can include portionsof the nucleic acid sequence of the SNS sodium channel or variantthereof useful for hybridizing with mRNA or DNA for use in assays todetect expression of the SNS sodium channel or localize its presence ona chromosome, such as the in situ hybridization assay described herein.

A conservative analogue is a protein sequence which retainssubstantially the same biological properties of the sodium channel butdiffers in sequences by one or more conservative amino acidsubstitutions. For the purposes of this document a conservative aminoacid substitution is a substitution whose probability of occuring innature is greater than ten times the probability of that substitutionoccuring by chance (as defined by the computational methods describedbyDayhoff et al, Atlas of Proteins Sequence and Structure, 1971, page95-96 and FIGS. 9-10).

A splice variant is a protein product of the same gene, generated byalternative splicing of mRNA, that contains additions or deletionswithin the coding region (Lewin B. (1995) Genes V Oxford UniversityPress, Oxford, England)

The nucleic acid sequences of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the protein of the present invention such as ahexa-histidine tag or a hemagglutinin (HA) tag.

The present invention further relates to nucleic acid sequences whichhybridize to the hereinabove-described sequences if there is at least50% and preferably 70% identity between the sequences. The presentinvention particularly relates to nucleic acid sequences which hybridizeunder stringent conditions to the hereinabove-described nucleic acidsequences. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences preferably the nucleic acidsequences which hybridize to the hereinabove described nucleic acidsequences encode proteins which retain substantially the same biologicalfunction or activity as the SNS sodium channel, however, nucleic acidsequences that have different properties are also within the scope ofthe present invention. Such sequences, while hybridizing with the abovedescribed nucleic acid sequences may encode a protein having diffferentproperties, such as sensitivity to tetrodotoxin which property is foundin the altered SNS sodium channel protein described herein.

In accordance with another aspect of the invention there is providedpurified mammalian sensory neuron sodium channel protein, wherein thesodium channel is insensitive to tetrodotoxin. Preferably the sodiumchannel of the invention is found in the neurons of the dorsal rootganglia or cranial ganglia, more preferably the neurons of the dorsalroot ganglia. The sodium channel protein may be derived from anymammalian species, preferably the rat or human sodium channel protein.The rat SNS sodium channel protein preferably has the deduced amino acidsequence shown in FIG. 1a (SEQ ID NO:2) or SEQ ID NO: 8, or the aminoacid sequence encoded by the deposited cDNA. Fragments, analogues,derivatives, and splice variants of the sodium channel specificallylocated in sensory neurons are also within the scope of the presentinvention.

The terms “fragment,” “derivative” and “analogue” when referring to theDRG sodium channel of the invention refers to a protein which retainssubstantially the same biological function or activity as such protein.Thus, an analogue includes a proprotein which can be activated bycleavage of the proprotein portion to produce an active mature protein.In addition, the present invention also includes derivatives wherein thebiological function or activity of the protein is significantly altered,including derivatives that are sensitive to tetrodotoxin.

The protein of the present invention may be a recombinant protein, anatural protein or a synthetic protein, preferably a recombinantprotein.

The fragment, derivative or analog of the SNS sodium channel proteinincludes, but is not limited to, (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituted group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the protein (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe mature protein, such as a leader or secretory sequence or a sequencewhich is employed for purification of the mature protein or a proproteinsequence, or (v) one in which one or more amino acids has/have beendeleted so that the protein is shorter than the full length protein.Variants of the rat SNS sodium channel are discussed hereinabove andshown in SEQ ID NO:4 and SEQ ID NO:6.

The proteins and nucleic acid sequences of the present invention arepreferably provided in an isolated form, and preferably are purified toat least 50% purity, more preferably about 75% purity, most preferablyabout 90% purity.

The terms “isolated” and/or “purified” mean that the material is removedfrom is original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring nucleic acidsequence or protein present in a living animal is not isolated orpurified, but the same nucleic acid sequence or DNA or protein,separated from some or all of the coexisting materials in the naturalsystem, is isolated or purified. Such nucleic acid sequence could bepart of a vector and/or such nucleic acid sequence or protein could bepart of a composition, and still be isolated or purified in that suchvector or composition is not part of its natural environment.

The present invention also provides vectors comprising a nucleic acidsequence of the present invention, and host cells transformed ortransfected with a nucleic of the invention.

The nucleic acid sequences of the present invention may be employed forproducing the SNS sodium channel protein or variant thereof byrecombinant techniques. Thus, for example, the nucleic acid sequence maybe included in any one of a variety of expression vehicles or cloningvehicles, in particular vectors or plasmids for expressing a protein.Such vectors include chromosomal, nonchromosomal and synthetic DNAsequences. Examples of suitable vectors include derivatives of SV40;bacterial plasmids; phage DNA; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, pseudorabies and baculovirus. However, anyother plasmid or vector may be used as long as it is replicable andviable in the host.

More particularly, the present invention also provides recombinantconstructs comprising one or more of the nucleic acid sequences asbroadly described above. The constructs comprise an expression vector,such as a plasmid or viral vector, into which a sequence of theinvention has been inserted, in a forward or reverse orientation. In apreferred aspect of this embodiment, the construct further comprises oneor more regulatory sequences, including, for example, a promoter,operably linked to the sequence. Large numbers of suitable vectors andpromoters are known to those of skill in the art, and are commerciallyavailable. The following vectors are provided by way of example.Bacterial: pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174,pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH461 (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL(Pharmacia) pcDNA 3.1 (Invitrogen, San Diego, Calif.), pEE14 (WO87/04462) and pREP8 (Invitrogen). Preferred vectors include pcDNA 3.1,pEE14 and pREP8. However, any other plasmid or vector may be used aslong as it is replicable and viable in the host.

As hereinabove indicated, the appropriate DNA sequence may be insertedinto the vector by a variety of procedures. In general, the DNA sequenceis inserted into appropriate restriction endonuclease sites byprocedures known in the art. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter and other promoters known to controlexpression of genes in prokaryotic or eukaryotic cells or their viruses.The expression vector may contain a ribosome binding site fortranslation initiation and transcription terminator. The vector may alsoinclude appropriate sequences for amplifying expression.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include LacI, LacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

Depending on the expression system employed in addition, the expressionvectors preferably contain a gene to provide a phenotypic trait forselection of transformed host cells such as dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, or such as tetracyclineor ampicillin resistance in E. coli.

Transcription of DNA encoding the protein of the present invention byhigher eukaryotes can be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100 to 270), a cytomegalovirus early promoter enhancer, apolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

Useful expression vectors for bacterial use may be constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseydomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, PKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,U.S.A.). These pBR322 “backbone” sections are combined with anappropriate promoter and the structural sequence to be expressed.

The sodium channel can be expressed in insect cells with the baculovirusexpression system which uses baculovirus such as Autographa Californicanuclear polyhydrosis virus (AcNPV) to produce large amounts of proteinin insect cells such as the Sf9 or 21 clonal lines derived fromSpodoptera frugiperda cells. See for example O'Reilly et al., (1992)Baculovirus Expression Vectors: A Laboratory Manual, Oxford UniversityPress.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In a further embodiment, the present invention provides host cellscapable of expressing a nucleic acid sequence of the invention. The hostcell can be, for example, a higher eukaryotic cell, such as a mammaliancell, a lower eukaryotic cell, such as a yeast cell, a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell may be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, electroporation (Davis, L., Dibner, M., Battey,I., Basic Methods in Molecular Biology, 1986) or any other method knownin the art.

Host cells are genetically engineered (transduced, transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the SNS sodium channel genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, and Salmonellatyphimurium; Streptomyces; fungal cells, such as yeast; insect cellssuch as Drosophila and Spodoptera fugiperda Sf9; animal cells such asCHO, COS or Bowes melanoma Ltk⁻- and Y1 adrenal carcinoma; plant cells,etc. The selection of an appropriate host is deemed to be within thescope of those skilled in the art based on the teachings herein.Preferred host cells include mammalian cell lines such as CHO-K11,COS-7; Y1 adrenal; carcinoma cells. More preferably, the host cells areCHO-K1 cells. Preferred host cells for transient expression of the SNSsodium channel include Xenopus laevis oocytes.

The sodium channel may be transiently expressed in Xeropus laevisoocytes. Cell-free translation systems can also be employed to producesuch proteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989).

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C 127, 3T3, CHO, CHO-K1, HeLa, HEK293, NIH 3T3 and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the proteins of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell-known to those skilled in the art.

The SNS sodium channel protein is recovered and purified fromrecombinant cell cultures by methods known in the art, includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, hydroxyapatite chromatographyand lectin chromatography. Protein refolding steps may be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The SNS sodium channel protein of the present invention may be naturallypurified products expressed from a high expressing cell line, or aproduct of chemical synthetic procedures, or produced by recombinanttechniques from a prokaryotic or eukaryotic host (for example, bybacterial, yeast, higher plant, insect and mammalian cells in culture).

The present invention also provides antibodies specific for the SNSsodium channel hereinabove defined. The term antibody as used hereinincludes all immunoglobulins and fragments thereof which containrecognition sites for antigenic determinants of proteins of the presentinvention. The antibodies of the present invention may be polyclonal orpreferably monoclonal, may be intact antibody molecules or fragmentscontaining the active binding region of the antibody, e.g. Fab or F(ab)₂and can be produced using techniques well established in the art [seee.g. R. A DeWeger et al; Immunological Rev., 62 p29-45 (1982)].

The proteins, their fragments or other derivatives, or analogs thereof,or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present also includes chimeric, single chainand humanized antibodies, as well as Fab fragments, or the product of anFab expression library. Various procedures known in the art may be usedfor the production of such antibodies and fragments.

Antibodies generated against the SNS sodium channel can be obtained bydirect injection of the polypeptide into an animal or by administeringthe protein to an animal, preferably a nonhuman. The antibody soobtained will then bind the protein itself. In this manner, even asequence encoding only a fragment of the protein can be used to generateantibodies binding the whole native protein. Such antibodies can then beused to locate the protein in tissue expressing that polypeptide. Forpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, 35al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss.,pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

The antibodies of the present invention may also be of interest inpurifying a protein of the present invention and accordingly there isprovided a method of purifying a protein of the present invention ashereinabove defined or any portion thereof or a metabolite or degrationproduct thereof which method comprises the use of an antibody of thepresent invention.

The purification method of the present invention may be effected by anyconvenient technique known in the art for example by providing theantibody on a support and contacting the antibody with a solutioncontaining the protein whereby the antibody binds to the protein of thepresent invention. The protein may be released from binding with theantibody by known methods for example by changing the ionic strength ofthe solution in contact with the complex of the protein/antibody.

The present invention also provides methods of identifying modulators ofthe sodium channel which is specifically located in sensory neuronscomprising contacting a test compound with the sodium channel anddetecting the activity of the sodium channel. Preferably, the methods ofidentifying modulators or screening assays employ transformed host cellsthat express the sodium channel. Typically, such assays will detectchanges in the activity of the sodium channel due to the test compound,thus identifying modulators of the sodium channel. Modulators of thesodium channel are useful in modulating the sensation of pain. Blockersof the sodium channel will prevent the transmission of impulses alongsensory neurons and thereby be useful in the treatment of acute, chronicor neuropathic pain.

The sodium channel can be used in a patch clamp or other type of assay,such as the assays disclosed herein in the examples, to identify smallmolecules, antibodies, peptides, proteins, or other types of compoundsthat inhibit, block, or otherwise interact with the sodium channel. Suchmodulators identified by the screening assays can then be used fortreatment of pain in mammals.

For example, host cells expressing the SNS sodium channel can beemployed in ion flux assays such as ²²Na+ ion flux and ¹⁴C guanidiniumion assays, as described in the examples and in the art, as well as theSFBI fluorescent sodium indicator assays as described in Levi et al.,(1994) J. Cardiovascular Electrophysiology 5:241-257. Host cellsexpressing the SNS sodium channel can also be employed in binding assayssuch as the 3H-batrachotoxin binding assay described in Sheldon et al.,(1986) Molecular Pharmacology 30:617-623; the 3H-saxitoxin assay asdescribed in Rogart et al (1983) Proc. Natl. Acad. Sci. USA80:1106-1110; and the scorpion toxin assay described in West et al.,(1992) Neuron 8:59-70. Additionally, the host cells expressing the SNSsodium channel can be used in electrophysiological assays using patchclamp or two electrode techniques. In general, a test compound is addedto the assay and its effect on sodium flux is determined or the testcompound's ability to competitively bind to the sodium channel isassessed. Test compounds having the desired effect on the SNS sodiumchannel are then selected. Modulators so selected can then be used fortreating pain as described above.

Complementary strands of the nucleotide sequences as hereinabove definedcan be used in gene therapy, such as disclosed in U.S. Pat. No.5,399,346. For example, the cDNA sequence or fragments thereof could beused in gene therapy strategies to down regulate the sodium channel.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a nucleic acid sequence to DNA or RNA. Forexample, the 5′ coding portion of the nucleic acid sequence that encodesthe sodium channel is used to design an antisense RNA oligonucleotide offrom about 10 to about 40 base pairs in length. A DNA oligonucleotide isdesigned to be complimentary to a region of the gene involved intranscription (triple helix-see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al, Science 241:456 (1988); and Deruau et al., Science251:1360 (1991)), thereby preventing transcription and the product ofthe sodium channel. The antisense RNA oligonucleotide hybridizes to themRNA in vivo and blocks translation of the mRNA into the sodium channel.Antisense oligonucleotides or an antisense construct driven by a strongconstituitive promoter expressed in the target sensory neurons would bedelivered either peripherally or to the spinal cord.

The regulatory regions controlling expression of the sodium channel genecould be used in gene therapy to control expression of a therapeuticconstruct in cells expressing the sodium channel.

Such regions would be isolated by using the cDNA as a probe to identifygenomic clones carrying the gene and also flanking sequence e.g.cosmids. Fragments of the cosmids containing intron or flanking sequencewould be used in a reporter gene assay in e.g. DRG cultures ortransgenic animals and genomic fragments carrying e.g. promoter,enhancer or LCR activity identified.

The invention will now be further described with reference to thefollowing examples:

EXAMPLE 1

Derivation of the Sequence of a Rat Dorsal Root Ganglia (DRG) SodiumChannel cDNA by Subtraction Hybridisation Methodology

1.1 cDNA Synthesis from DRG-derived poly-A+ RNA

Dorsal root ganglia (DRG) from all spinal levels of neonatalSprague-Dawley male and female rats were frozen in liquid nitrogen. RNAis extracted using guanidine isothiocyanate and phenol/chloroformextraction (Chomczynski and Sacchi 1987 Anal Biochem 162, 156-159).

Total RNA isolation—the nerve tissue is homogenised using a Polytronhomogeniser in 1 ml extraction buffer (23.6 g guanidiniumisothiocyanate, 5 ml of 250 mM sodium citrate (pH 7.0) made up to 50 mlwith distilled water. To this is added 2.5 ml 10% sarcosyl and 0.36 mlβ-mercaptoethanol). 0.1 ml of 2M sodium acetate (pH 4.0) is addedfollowed by 1 ml phenol. After mixing, 0.2 ml chloroform is added andthis is shaken vigorously and placed on ice for 5 minutes. This is thencentrifuged at 12,000 revolutions per minute (rpm) for 30 minutes at 4°C. The aqueous phase is transferred to a fresh tube, 1 ml of isopropanolis added and this is left at −20° C. for an hour followed bycentrifuging at 12000 rpm for 30 minutes at 4° C. The pellet isdissolved in 0.1 ml extraction buffer and is again extracted withisopropanol. The resulting pellet is washed with 70% ethanol and isresuspended in diethyl pyrocarbonate (DEPC)-treated water. 0.3M sodiumacetate (pH5.2) and 2 volumes of ethanol are added and the mixture isplaced at −20° C. for 1 hour. The RNA is precipitated, washed again with70% ethanol and resuspended in DEPC-treated water. The optical densityis measured at 260 nanometres (nm) to calculate the yield of total RNA.Poly A+ RNA is isolated from the total RNA by oligo-dT cellulosechromatography (Aviv and Leder 1972 Proc Natl Acad Sci 69,1408-1411).The following procedures are carried out at 4° C. as far as is possible.Oligo-dT cellulose (Sigma) is prepared by treatment with 0.1M sodiumhydroxide for 5 minutes. The oligo-dT resin is poured into a column andis neutralised by washing with neutralising buffer (0.5 M potassiumchloride, 0.01M Tris (Trizma base—Sigma-Tris(hydroxymethyl)aminomethane)(pH 7.5). The RNA solution is adjusted to 0.5M potassium chloride, 0.01MTris (pH7.5) and is applied to the top of the column. The first columneluate is re-applied to the column to ensure sticking of the mRNA to theoligo-dT in the column. The column is then washed with 70 ml ofneutralising buffer and the polyA+ RNA is eluted with 6 ml 0.01M Tris(pH7.5) and 1 ml fractions are collected. The poly A+ RNA is usually infractions 2 to 5 and this is checked by measuring the optical density at260 nm. These fractions are pooled and ethanol precipitated overnight at−70° C., washed in 70% ethanol and then redissolved in deionised waterat a concentration of 1 mg/ml.

First strand CDNA was generated using 0.5 mg DRG poly A+ mRNA,ligo-dT/Not-I primer adapters and SuperScript reverse transcriptase(Gibco-BRL) using methodology as described in example 2. One half of thecDNA was labelled by including 2 MBq ³²P dCTP (Amersham) in the reversetranscriptase reaction. Labelled cDNA is separated from unincorporatednucleotides on Nick columns (Sephadex G50-Pharmacia).

1.2 Enrichment of DRG-specific cDNA Using Subtraction Hybridisation

Poly A+ RNA from various tissues (10 μg) is incubated with 10 μgphotoactivatable biotin (Clontech) in a total volume of 15 μl andirradiated at 4° C. for 30 minutes with a 250 watt sunlamp. Thephotobiotin is removed by extraction with butanol, and the cDNAco-precipitated with the biotinylated RNA without carrier RNA (Sive andSt. John 1988 Nuc Ac Res 16,10937).

Hybridisation is carried out at 58° C. for 40 hours in 20% formamide, 50mM 3-(N-morpholino)propanesulphonic acid (MOPS) (pH 7.6), 0.2% sodiumdodecyl sulphate (SDS), 0.5M sodium chloride, 5 mMethylenediaminetetraacetate (EDTA—Sigma). The total reaction volume is 5μl and the reaction is carried out under mineral oil, after an initialdenaturation step of 2 minutes at 95° C. 100 μl 50 mM MOPS (pH 7.4),0.5M sodium chloride, 5 mM EDTA containing 20 units of streptavidin(BRL) is then added to the reaction mixture at room temperature, and theaqueous phase retained after two phenol/chloroform extraction steps.After sequential hybridisation of the cDNA from Example 1.1 withbiotinylated mRNA from liver and kidney, followed by cortex andcerebellum, a 80-fold concentration of DRG-specific transcripts isachieved.

One third of the 1-2 ng of residual cDNA is then G-tailed with terminaldeoxynucleotide transferase at 37° C. for 30 minutes. The polymerasechain reaction is used to amplify the cDNA using an oligo-dT-Not-Iprimer adapter and oligo-dC primers starting with the sequenceAATTCCGA(C)₁₀. Amplification is carried out using 2 cycles of 95° C. for1 min, 45° C. for 1 min, 72° C. for 5 min, followed by 2 cycles of 95°C. for 1 minute, 58° C. for 1 minute and 72° C. for 5 minutes. Theresulting products are then separated on a 2% Nu-sieve agarose gel, andmaterial running at a size of greater than 0.5 kilobase pairs (kb) iseluted and further amplified with 6 cycles of 95° C. for 1 minute, 58°C. for 1 minute and 72° C. for 5 minutes. This material is furtherseparated on a 2% Nu-sieve agarose gel, and the material running from 6kb on the gel is eluted and further amplified using the same PCRconditions for 27 cycles. The amplified DNA derived from this highmolecular weight region is then further fractionated on a 2% Nu-Sievegel, and cDNA from 0.5 to 1.5 kb, and from 1.5 to 5 kb pooled.

1.3. Library Construction

10 μg of the bacteriophage vector lambda-zap II (Stratagene) isrestriction digested with NotI and EcoRI in high salt buffer overnightat 37° C. followed by dephosphorylation using 1 unit of calf intestinalphosphatase (Promega) for 30 minutes at 37° C. in 10 mM Tris.HCl(pH9.5), 1 mM spermidine, 0.1 mM EDTA. DRG cDNA is digested with Klenowenzyme in the presence of dGTP and dCTP to construct an EcoRI site fromthe oligo-dC primer (see above) at the 5′ end of the cDNA, and cut withNotI for directional cloning. The cDNA is ligated into the cloningvector bacteriophage lambda-zap II for 16 hours at 12° C. Recombinantphage DNA is then packaged into infective phage using Gigapack gold(Stratagene) and protocols specified by the suppliers. 0.1% of thepackaged DNA is used to infect E.coli BB4 cells which are plated out tocalculate the number of independent clones generated.

1.4 Differential Screening

The library is plated at a low density (10³ clones/12×12 cm² dish) andscreened using three sets of ³²P-labelled cDNA probes and multiplefilter lifts. Replica filters are made by laying them onto the platedlibrary plates, briefly drying them and then laying onto fresh agarplates to increase the quantity of phage and the subsequenthybridisation signals of lifts taken from them. The probes are derivedfrom: a) cortex and cerebellum poly (A)+ RNA, b) DRG poly (A)+ RNA, andc) subtracted cDNA from DRG. The two mRNA probes are labelled with ³²PdCTP using a reaction mixture containing 2-5 μg RNA, 50 μl 5×RT buffer,25 μl 0.1M dithiothreitol (DTT), 12.5 μl 10 mM dATP, dGTP, dCTP, 30 pMoligo-dT, 75 μl ³²P-dCTP (30 MBq; Amersham), 25 μl 100 μM dCTP, 2 μlRNasin (2 units/μl) and 2 μl SuperScript reverse transcriptase(GibcoBRL) in a final volume of 250 μl. The reaction is incubated at 39°C. for 60 minutes, and the RNA subsequently destroyed by adding 250 μlwater, 55 μl 1M NaOH, and incubating at 70° C. for 20 minutes. Thereaction mixture is neutralised with acidified Tris base (pH 2.0) andprecipitated with carrier tRNA (Boehringer) with isopropanol. Thesubtracted and amplified double-stranded DRG cDNA is random-primelabelled with ³²P dATP (Gibco multiprime kit). Replica filters are thenprehybridised for 4 hours at 68° C. in hybridisation buffer.Hybridisation was carried out for 20 hours at 68° C. in 4×SSC (20×SSCconsists of 175.3 g of sodium chloride and 88.2 g of sodium citrate in800 ml of distilled water. The pH is adjusted to 7.0 with 10N sodiumhydroxide and this is made to 1 liter with distilled water), 5×Denhardtssolution containing 150 μg/ml salmon sperm DNA, 20 μg/ml poly-U, 20μg/ml poly-C, 0.5% SDS (Sigma), 5 mM EDTA. The filters are brieflywashed in 2×SSC at room temperature, then twice with 2×SSC with 0.5% SDSat 68° C. for 15 minutes, followed by a 20 minute wash in 0.5% SDS,0.2×SSC at 68° C. The filters are autoradiographed for up to I week onKodak X-omat film. Plaques that hybridise with DRG probes but not cortexand cerebellum probes are picked, phage DNA prepared and the clonedinserts released for subcloning into pBluescript (Stratagene).

The positive plaques are picked by lining up the autoradiogram with theplate using orientation marks and taking a plug from the platecorresponding to the positive hybridisation signal. The phage is elutedfrom the plug in 0.5 ml phage dilution buffer (10 mM Tris chloride(pH7.5) 10 mM magnesium sulphate) and the phage re-infected into E.coliBB4 and replated at a density of 200 to 1000 plaques/150 mm plate as asecondary purification step to ensure purity of the clones. The positivesecondaries are then picked as described previously. In order tosub-clone the insert DNA from the positive recombinant phage, they needto be amplified. This is accomplished by plate lysis where the phagetotally lyse the E.coli BB4. 0.2 ml of phage suspension is mixed with0.1 ml of an overnight culture of E.coli. This is added to 2.5 ml of topagar (16 g bacto-tryptone 10 g bacto-yeast extract, 5 g sodium chloride,7 g bacto-agar in 900 mls distilled water) and plated onto 9 cm² agarplates. These are incubated overnight at 37° C. 5 ml of phage dilutionbuffer is then added to the plates and is incubated overnight at 4° C.or for 4 hours with gentle scraping at room temperature. Thephage-containing buffer is then recovered, 0.1 ml chloroform is addedand this phage stock is titrated as above and stored at 4° C. Phage DNAis prepared by first infecting 10¹⁰ E.coli B44 with 10⁹ plaque formingunits (pfus) of phage in 3 ml of phage dilution buffer and shaking at37° C. for 20 minutes. The infected bacteria are added to 400 ml of Lbroth (1.6% bactotryptone, 0.5% (w/v) Bacto yeast extract, 0.5% (w/v)magnesium sulphate) with vigorous shaking at 37° C. for 9 hours. Whenlysis has occurred, 10 ml of chloroform is added and shaking iscontinued for a further 30 minutes. The culture is then cooled to roomtemperature and pancreatic RNAase and DNAase are added to 1 ug/ml for 40minutes. Sodium chloride is then added to 1M and is dissolved byswirling on ice. After centrifuging at 8000 rpm for 10 minutes thesupernatant is recovered. Polyethylene glycol (PEG 6000) is added to 10%w/v and is dissolved by stirring whilst on ice for 2 hours. Aftercentrifuging for 8000 rpm for 10 minutes at 4° C. the pellet isresuspended in 8 ml of phage dilution buffer. This is extracted with anequal volume of phenol/chloroform followed by purification on a caesiumchloride gradient (0.675 g/ml caesium chloride—24 hours at 38000 rpm at4° C.). The opaque phage band is removed from the centrifugation tubeand dialysed against 10 mM sodium chloride, 50 mM Tris (pH8.0), 10 mMmagnesium chloride for 2 hours. EDTA is then added to 20 mM, proteinaseK to 50 μg/ml and SDS to 0.5% and is incubated at 65° C. for 1 hour.After dialysis overnight against TE pure phage DNA results. The clonedinsert is digested from the purified phage DNA using restriction enzymesas previously described. Each phage insert is then ligated into aplasmid vector e.g. pBluescript—Clontech using a ligation reaction aspreviously described.

Clone Characterisation

The plasmids are cross hybridised with each other. Unique clones arefurther analysed by Northern blotting and sequencing. The clone/sshowing transcript sizes and sequence comparable with sodium channelsare then used as hybridisation probes to screen a neonatal rat DRG oligodT-primed full length cDNA library to derive full length cDNA clonesusing methodology as described above and in example 2. Biologicalactivity of the rat DRG sodium channel is confirmed as in examples 4 and7 below.

EXAMPLE 2

Homology Cloning of the Human cDNA Homologous to the Rat DRG SodiumChannel cDNA (SNS-B)

2.1. Isolation of Human Ganglia Total RNA

The starting material for the derivation of the human cDNA homologue ofthe rat DRG sodium channel cDNA is isolated human dorsal root ganglia ortrigeminal ganglia or other cranial ganglia from post-mortem humanmaterial or foetuses. Total ribonucleic acid (RNA) is isolated from thehuman neural tissue by extraction in guanidinium isothiocyanate(Chomczynski and Sacchi 1987 Anal Biochem 162,156-159) as described inexample 1.

2.2 Determination of the Transcript Size of the Human Homologue of theRat DRG Sodium Channel cDNA (SNS-B)

Human dorsal root ganglia total RNA is electrophoretically separated ina 1% (w/v) agarose gel containing a suitable denaturing agent e.g.formaldehyde (Lehrach et al 1977 Biochemistry 16,4743; Goldberg 1980Proc Natl Acad Sci 77,5794; Seed 1982 in Genetic engineering: principlesand methods (ed J K Setlow and A Hollaender) vol 4 p91 Plenum PublishingNew York) or glyoxal/DMSO (McMaster G K and Carmichael G G 1977 ProcNatl Acad Sci 74,4835), followed by transfer of the RNA to a suitablemembrane (e.g. nitrocellulose). The immobilised RNA is then hybridisedto radioactive (or other suitable detection label) probes consisting ofportions of the rat sodium channel cDNA sequence (see below). Afterwashing of the membrane to remove non-hybridised probe, the hybridisedprobe is visualised using a suitable detection system (e.g.autoradiography for ³²P labelled probes) thus revealing the size of thehuman homologous mRNA molecule. Specifically, 20-30 μg total RNA fromneonatal rat tissues are separated on 1.2% agarose-formaldehyde gels,and capillary blotted onto Hybond-N (Amersham) (Ninkina et al. 1993 NucAc Res 21,3175-3182). The amounts of RNA on the blot are roughlyequivalent, as judged by ethidium bromide staining of ribosomal RNA orby hybridisation with the ubiquitously expressed L-27 ribosomal proteintranscripts (Le Beau et al. 1991 Nuc Ac Res 19,1337). Each Northern blotcontains human DRG, cortex, cerebellum, liver kidney, spleen and heartRNA. Probes (50 ng) are labelled with ³²P-dATP (Amersham) by randompriming. Filters are prehybridised in 50% formaldehyde 5×SSC containing0.5% SDS, 5×Denhardts solution (50×Denhardts contains 5 g of Ficoll(Type 400, Pharmacia), 5 g of polyvinylpyrrolidone, 5 g of bovine serumalbumin (Fraction V, Sigma) and water to 500 ml), 100 μg/ml boiledsalmon sperm DNA, 10 μg/ml poly-U and 10 μg/ml poly-C at 45° C. for 6hours. After 36 hours hybridisation in the same conditions, the filtersare briefly washed in 2×SSC at room temperature, then twice with 2×SSCwith 0.5% SDS at 68° C. for 15 minutes, followed by a 20 minute wash in0.5% SDS, 0.2×SSC at 68° C. The filters are autoradiographed for up to 1week on Kodak X-omat film. The transcript size is calculated from thesignal from the gel in comparison with gel molecular weight standardmarkers.

2.3 Production of a Human DRG cDNA Library

In order to produce a representative cDNA library from the human dorsalroot ganglia messenger RNA (poly A+ mRNA) is first isolated from thetotal RNA pool using oligo-dT cellulose chromatography (Aviv and Leder1972 Proc Natl Acad Sci 69,1408-1411) using methodology described inexample 1. Synthesis of the first strand of cDNA from the polyA+ RNAuses the enzyme RNA-dependent DNA polymerase (reverse transcriptase) tocatalyse the reaction. The most commonly used method of second strandcDNA synthesis uses the product of first strand synthesis, a cDNA:mRNAhybrid, as a template for priming the second strand synthesis. (Gublerand Hoffman 1983 Gene 25,263)).

2.3.1. First Strand cDNA Synthesis

20 μg of human DRG polyA+ RNA is pre-treated to destroy secondarystructure which may inhibit first strand cDNA synthesis. 20 μg of polyA+RNA, 1 μl 1M Tris (pH7.5) are made up to a volume of 100 μl withdistilled water. This is incubated at 90° C. for 2 minutes followed bycooling on ice. 4.8 μl of 100 mM methyl mercury is then added for 10minutes at room temperature. 10 μl of 0.7M β-mercaptoethanol and 100units of human placental RNAase inhibitor are then added for 5 minutesat room temperature. The first strand synthesis reaction consists of 8μl 20 mM dATP, 5 μl 20 mM dCTP, 8 μl 20 mM dGTP 8 μl 20 mM dTTP, 10 μl 1mg/ml oligo-dT (12-18), 20 μl 1M Tris (pH 8.3) (at 45° C.), 8 μl 3Mpotassium chloride, 3.3 μl 0.5M magnesium chloride, 3 μl a³²P dCTP, 100units Superscript II reverse transcriptase (GibcoBRL) made up to 200 μlwith distilled water. This reaction mixture is incubated at 45° C. for45 minutes after which another 50 units of Superscript reversetranscriptase is added and incubated for a further 30 minutes at 45° C.EDTA is then added to 10 mM to terminate the reaction and aphenol/chloroform extraction is carried out. The DNA is thenprecipitated using ammonium acetate (freezing in dry ice/ethanol beforecentrifuging), washed with 70% ethanol and resuspended in 50 mldistilled water. The size of the single stranded DNA is assessed byelectrophoretically separating it out on an agarose gel (1% w/v) andautoradiographing the result against markers.

2.3.2 Second Strand Synthesis

The second strand synthesis reaction mixture consists of 0.5 μg humanDRG single stranded DNA, 2 μl 1M Tris (pH7.5), 1 μl 0.5M magnesiumchloride, 3.33 μl 3M potassium chloride, 2 μl 0.5M ammonium sulphate,1.5 μl 10 mM βnicotinamide adenine dinucleotide (NAD), 4 μl of each ofthe 1 mM dNTPs, 5 μl 1 mg/ml bovine serum albumin (BSA), 1 unitRNAase-H, 25 units Klenow polymerase all made up to 100 μl withdistilled water. This is incubated at 12° C. for 1 hour and then at 20°C. for 1 hour. The reaction is stopped by addition of EDTA to 20 mMfollowed by a phenol/chloroform extraction. The DNA is ethanolprecipitated (−70° C. overnight) and is then washed with 70% ethanolfollowed by resuspension in 20 μl distilled water. Size is checked bygel electrophoresis and autoradiography.

2.3.3 Double Stranded cDNA end Repair

In order to add linkers to the end of the cDNA molecules for subsequentcloning, the ends must first be repaired. The human DRG cDNA is treatedwith 500 units/ml of S1 nuclease in 0.25M sodium chloride, 1 mM zincsulphate, 50 mM sodium acetate (pH4.5). Incubation is at 30° C. for 40minutes followed by neutralisation with Tris (pH 8.0) to 0.2M. The DNAis again ethanol precipitated, washed in 70% ethanol and resuspended in20 μl distilled water. The size is again checked to ensure that S1nuclease digestion has not radically reduced the average DNA fragmentsize. The repair reaction consists of 19 μl cDNA, 3 μl 10×T4 polymerasebuffer (0.33M Tris acetate (pH7.9), 0.66M potassium acetate, 0.1Mmagnesium acetate, 1 mg/ml BSA and 5 mM DTT), 2 μl of each dNTP at 2 mM,2 μl T4 polymerase and 4 μl distilled water. This is incubated at 37° C.for 30 minutes followed by addition of 1 μl Klenow polymerase for 1 hourat room temperature. The DNA is then ethanol precipitated, washed in 70%ethanol and resuspended in 5 μl distilled water. In order to protectnaturally occurring restriction sites within the cDNA from beingcleaved, the cDNA is treated with a methylase before the addition oflinkers. The reaction mixture consists of 5 μl human DRG double strandedDNA, 1 μl S-adenosylmethionine, 2 μl 1 mg/ml BSA, 2 μl 5×methylasebuffer (0.5M Tris (pH8.0), 5 mM EDTA), 0.2 μl EcoRI methylase (NEB).This is incubated at 37° C. for 20 minutes followed by phenolextraction, ethanol precipitation washing with 70% ethanol andresuspension in 20 μl distilled water.

2.3.4. Addition of Linkers to cDNA

EcoRI linkers are ligated to the cDNA molecules to facilitate cloninginto lambda vectors. The ligation reaction mixture consists of 1 μl 10×ligation buffer (0.5M Tris chloride (pH7.5), 0.1M magnesium chloride and0.05M DTT), 1 μl 10 mM ATP, 100 ng cDNA, 5 μg EcoRI linkers, 1 unit T4DNA ligase, distilled water to 10 μl. The reaction is incubated at 37°C. for 1 hour, followed by addition of 6 more units of T4 ligase and afurther incubation overnight at 15° C. The ligated samples are ethanolprecipitated, washed in 70% ethanol and resuspended in 10 μl distilledwater. The cDNA is then digested with EcoRI to cleave any linkerconcatamers formed in the ligation process. This restriction digestionreaction contains 10 μl cDNA, 2 μl high salt buffer (10 mM magnesiumchloride, 50 mM Tris chloride (pH7.5), 1 mM DTT, 100 mM sodiumchloride), 2 μl EcoRI (10 units/μl—NEB) and distilled water to 20 μl.The digestion is carried out for 3 hours. The ligation and digestionsteps are monitored using gel elecrophoresis to monitor the size of theproducts.

2.3.5 Size Fractionation of cDNA

In order to assure that the library is not swamped with short cDNAmolecules and to remove linker molecules a column purification iscarried out. A 1 ml Sepharose 4B column is made in a 1 ml plasticpipette plugged with a small piece of glass wool. This is equilibratedwith 0.1M sodium chloride in TE. The cDNA is loaded onto the column and1 drop fractions are collected. 2 μl aliquots of each fraction areanalysed by gel electrophoresis and autoradiography to determine thesizes of the cDNA in each fraction. Fractions containing cDNA of about800 base pairs and above are pooled and purified by ethanolprecipitation and resuspending in 10 μl distilled water.

2.3.6 Cloning of cDNA into Bacteriophage Vector

Bacteriophage vectors designed for the cloning and propagation of cDNAare provided ready-digested with EcoRI and with phosphatased ends fromcommercial sources (e.g. lambda gt10 from Stratagene). The preparedsubtracted cDNA is ligated into lambda gt10 using a ligation rectionconsisting of ligase buffer and T4 DNA ligase (New England Biolabs) asdescribed elsewhere in this document.

2.4 Labelling of cDNA Fragments (Probes) for Library Screening

The 3′ untranslated region of the rat DRG sodium channel cDNA clone(SNS-B) is subcloned using appropriate restriction enzymes into aplasmid vector e.g. pBluescript—Stratagene. The cDNA insert which is toform the labelled probe is released from the vector via digestion withappropriate restriction enzymes and the insert is separated from thevector via electrophoresis in a 1% (w/v) agarose gel. After removal ofthe separated insert from the agarose gel and purification it islabelled by standard techniques such as random priming andpolymerisation (Feinberg and Vogelstein 1983 Anal Biochem 132,6) or nicktranslation (Rigby et al 1977 J Mol Biol 113,237) with ³²P orDIG-labelled nucleotides. Alternatively, if the probe cDNA insert iscloned into a vector containing strong bacteriophage promoters to whichDNA-dependant RNA polymerases bind (SP6, T3 or T7 polymerases),synthetic cRNA is produced by in vitro transcription which incorporates³²P or digoxygenin nucleotides. Other regions of the rat DRG sodiumchannel cDNA can also be used as probes in a similar fashion for cDNAlibrary screening or Northern blot analysis. Specifically, a probe ismade using a kit such as the Pharmacia oligo labelling kit. This willradioactively label the rat DRG sodium channel cDNA fragment. 50 ng ofdenatured DNA (place in boiling waterbath for 5 minutes), 3 μl of³²PdCTP (Amersham) and 10 μl reagent mix is made up to 49 μl withdistilled water. 1 μl of Klenow fragment is added and the mixture isincubated at 37° C. for one hour. To remove unincorporated nucleotides,the reaction mixture is applied to a Nick column (SephadexG50-Pharmacia) followed by 400 μl of TE (10 mM Tris chloride (pH7.4) 1mM EDTA (pH8.0)). Another 400 μl of TE is added and the eluate iscollected. This contains the labelled DNA to be used as a hybridisationprobe.

2.5 cDNA Library Screening

In order to detect recombinants containing human homologues of the ratDRG sodium channel the human DRG cDNA library is screened using moderatestringency hybridisation washes (50-60° C., 5×SSC, 30 minutes), usingradiolabelled or other labelled DNA or cRNA probes derived from the 3′untranslated region as described above. Libraries are screened usingstandard methodologies involving the production of nitrocellulose ornylon membrane replicas of DNA from recombinant plaques formed on agarplates (Benton et al 1977 Science 196;180). These are then hybridised tosingle stranded nucleic acid probes (see above). Moderate stringencywashes are carried out (see wash conditions for Northern analysis insection 2.2). Plaques which are positive on duplicate filters (i.e. notartefacts or background) are then purified by one or more rounds ofreplating after dilution to separate the colonies and furtherhybridisation screening. Resulting positive plaques are purified, DNA isextracted and the insert sizes of these clones is examined. The clonesare cross-hybridised to each other using standard techniques (Sambrooket al 1989 Molecular Cloning Second Edition Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and distinct positive clonesidentified. Detailed protocols for cDNA library screening are given inexample 1.

2.6 Derivation of a Full-length Clone of the Human Homologue of the RatDRG Sodium Channel cDNA

Overlapping positive clones from above are identified bycross-hybridisation. They are then restriction mapped to identify theircommon portions and restriction fragments representing the separateportions from the overlapping clones are ligated together using standardcloning techniques (Sambrook et al 1989 Molecular Cloning Second EditionCold Spring Harbor Laboratory Press). For example, the most 5′ fragmentwill contain any 5′ untranslated sequence, the start codon ATG and 5′coding sequence. The most 3′ clone will contain the most 3′ codingsequence, a stop codon and any 3′ untranslated sequence, a poly Aconsensus sequence and possibly a poly A run. Thus a recombinantmolecule is generated which contains the full cDNA sequence of the humanhomologue of the rat DRG sodium channel cDNA. If overlapping clones donot produce sufficient fragments to assemble a full length cDNA clone,the full length oligo dT-primed human DRG library is re-screened toisolate a full length clone. Alternatively, a full length clone isderived directly from the library screening.

2.7 Characterisation of the Human Homologue Full-length Clone

The cDNA sequence from the full-length clone is used as a probe inNorthern blot analysis to detect the messenger RNA size in human tissuefor comparison with the rat messenger RNA size (see sections 1.1 and 2.2for methodology).

Confirmation of biological activity of the cloned cDNA is carried outvia in vitro translation of the human sodium channel mRNA and itsexpression in Xenopus oocytes in an analogous manner to that for the ratDRG-specific TTXi resistant sodium channel as described in examples 4and 7.

cDNA sequences which are shown to have activity as defined above arecompletely sequenced using dideoxy-mediated chain termination sequencingprotocols (Sanger et al 1977 Proc Natl Acad Sci 74,5463).

EXAMPLE 3

Polymerase Chain Reaction (PCR) Approaches to Clone the Human DRG SodiumChannels Using DNA Sequence Derived from the Rat DRG Sodium Channel cDNAClone

Total RNA and poly A+ RNA is isolated from human dorsal root ganglia ortrigeminal ganglia or other cranial ganglia from post-mortem humanmaterial or foetuses as described in example 2 above.

Random primers are hybridised to the RNA followed by polymerisation withMMLV reverse transcriptase to generate single stranded cDNA from theextracted human RNA.

Using degenerate PCR primers derived from relatively conserved regionsof the known voltage-gated sodium channels (FIG. 2), amplify the cDNAusing the polymerase chain reaction (Saiki et al 1985 Science 230,1350).It is appreciated by those skilled in the art that there are manyvariables which can be manipulated in a PCR reaction to derive thehomologous sequences required. These include but are not limited tovarying cycle and step temperatures, cycle and step times, number ofcycles, thermostable polymerase, Mg2+ concentration. It is alsoappreciated that greater specificity can be gained by a second round ofamplification utilising one or more nested primers derived from furtherconserved sequence from the sodium channels.

Specifically, the above can be accomplished in the following manner. Thefirst strand cDNA reaction consists of 1 μg of total RNA made up to 13μl with DEPC-treated water and 1 μl of 0.5 μg/μl oligo(dT). This isheated to 70° C. for 10 minutes and then incubated on ice for 1 minute.The following is then added: 2 μl of 10 × synthesis buffer (200 mM Trischloride, 500 mM potassium chloride, 25 mM magnesium chloride, 1 μg/mlBSA), 2 μl of 0.1M DTT, 1 μl of 200 U/μl Superscript ReverseTranscriptase (Gibco BRL). This is incubated at room temperature for 10minutes then at 42° C. for 50 minutes. The reaction is then terminatedby incubating for 15 minutes at 70° C. 1 μl of E.coli RNase H (2 U/μl)is added to the tube which is then incubated for 20 minutes at 37° C.

The PCR reaction is set up in a 0.5 ml thin-walled Eppendorf tube. Thefollowing reagents are added: 10 μl 10× PCR buffer, 1 μl cDNA,16 μldNTP's (25 μl of 100 μM dATP,dCTP, dCTP and dGTP into 900 μl steriledistilled water), 7 μl of 25 mM magnesium chloride, 1 μl of Taq DNApolymerase (Amplitaq Perkin-Elmer)plus sterile distilled water to 94 μl.

To each reaction tube a wax PCR bead is added (Perkin-Elmer) and thetube placed in a 70° C. hot block for 1 minute. The tubes are allowed tocool until the wax sets and 3 μl of each primer (33 pM/μl) are addedabove the wax. The tubes are placed in a thermal cycler (Perkin-Elmer)and the following 3-step program used after an initial 94° C. for 5minutes; 92° C. for 2 minutes, 55° C. for 2 minutes, 72° C. for 2minutes for 35 cycles. A final polymerisation step is added at 72° C.for 10 minutes. The reaction products are then run on a 1% agarose gelto assess the size of the products. In addition, control reactions areperformed alongside the samples. These should be: 1) all componentswithout cDNA (negative control) and 2) all reaction components withprimers for constitutively expressed product e.g. α-actin or HPRT.

The products of the PCR reactions are examined on 0.8%-1.2% (w/v)agarose gels. Bands on the gel (visualised by staining with ethidiumbromide and viewing under UV light) representing amplification productsof the approximate predicted size were then cut from the gel and the DNApurified. Further bands of interest are also identified by Southern blotanalysis of the amplification products and probing of the resultingfilters with labelled primers from further conserved regions e.g. thoseused for secondary amplification.

The resulting DNA is ligated into suitable vectors such as, but notlimited to, pCR II (Invitrogen) or pGemT. Clones are then sequenced toidentify those containing sequence with similarity to the rat DRG sodiumchannel sequence (SNS-B).

Clone Analysis

Candidate clones from above are used to screen a human cDNA DRG libraryconstructed using methods described in example 2. If a full length cloneis not identified, positive overlapping clones which code for the fulllength human cDNA homologue are identified and a full length clone isthen assembled as described in example 1. Biological activity is thenconfirmed as described in examples 4 and 7.

EXAMPLE 4

In vitro Translation of Rat and Human DRG Sodium Channel in Xenopuslaevis Oocytes

In order to demonstrate the biological activity of the protein coded forby the rat DRG sodium channel cDNA sequence (SNS-B) and its humanhomologue the complete double-stranded cDNA coding sequences are ligatedinto in vitro transcription vectors (including but not limited to thepGEM series, Promega) using one or more of the available restrictionenzyme sites such that the cDNAs are inserted in the correctorientation. The constructs are then used to transform bacteria andconstructs with the correct sequence in the correct orientation areidentified via diagnostic restriction enzyme analysis anddideoxy-mediated chain termination DNA sequencing (Sanger et al 1977Proc Natl Acad Sci 74,5463).

These constructs are then linearised at a restriction site downstream ofthe coding sequence and the linearised and purified plasmids are thenutilised as a template for in vitro transcription. Sufficient quantitiesof synthetic mRNA are produced via in vitro transcription of the clonedDNA using a DNA-dependent RNA polymerase from a bacteriophage thatrecognises a bacteriophage promoter found in the cloning vector.Examples of such polymerases include (but are not limited to) T3, T7 andSP6 RNA polymerase.

A variation on the above method is the synthesis of mRNA containing a 5′terminal cap structure (7-methylguanosine) to increase its stability andenhance its translation efficiency (Nielson and Shapiro 1986 Nuc Ac Res14,5936). This is accomplished by the addition of 7-methylguanosine tothe reaction mixture used for synthetic mRNA synthesis. The capstructure is incorporated into the 5′ end of the transcripts aspolymerisation occurs. Kits are available to facilitate this processe.g. mCAP RNA Capping Kit—Stratagene).

The synthetic RNA produced from the in vitro transcription is isolatedand purified. It is then translated via microinjection into Xenopuslaevis oocytes. 50 nls of 1 mg/ml synthetic RNA is micro-injected intostage 5 or stage 6 oocytes according to methods established in theliterature (Gurdon et al (1983) Methods in Enzymol 101,370). Afterincubation to allow translation of the mRNAs the oocytes are analysedfor expression of the DRG sodium channels via electrophysiological orother methods as described in example 7.

A further method for expression of functional sodium channels involvesthe nuclear injection of a Xenopus oocyte protein expression vector suchas pOEV (Pfaff et al., Anal. Biochem. 188, 192-195 (1990)) which allowscloned DNA to be transcribed and translated directly in the oocyte.Since proteins translated in oocytes are post-translationally modifiedaccording to conserved eukaryotic signals, these cells offer aconvenient system for performing structural and functional analyses ofcloned genes. pOEV can be used for direct analysis of proteins encodedby cloned cDNAs without preparing mRNA in vitro, simplifying existingprotocols for translating proteins in oocytes with a very hightranslational yield. Transcription of the vector in oocytes is driven bythe promoter for the TFIIIA gene, which can generate 1-2 ng (per oocytewithin 2 days) of stable mRNA template for translation. The vector alsocontains SP6 and T7 promoters for in vitro transcription to make mRNAand hybridization probes. DNA clones encoding SNS channel transcriptsare injected into oocyte nuclei and protein accumulated in the cell overa 2- to 10-day period. The presence of functional protein is thenassessed using twin electrode voltage clamp as described in example 7.

EXAMPLE 5

Expression of Rat and Human DRG Sodium Channel in Mammalian Cells

In order to be able to establish a mammalian cell expression systemcapable of producing the sodium channel in a stable bloactive manner,constructs have to be first generated consisting of the cDNA of thechannel in the correct vectors suitable for the cell system in which itis desired to express the protein. There are available a range ofvectors containing strong promoters which drive expression in mammaliancells.

i/Transient Expression

In order to determine rapidly the bioactivity of a given cDNA it can beintroduced directly into cells and resulting protein activity assayed48-72 hours later. Although this does not result in a cell line which isstably expressing the protein of interest it does give a quick answer asto the biological activity of the molecule. Specifically, the cDNArepresenting the human or rat DRG sodium channel is ligated intoappropriate vectors (including but not limited to pRc/RSV, pRc/CMV,pcDNA1 (Invitrogen)) using appropriate restriction enzymes such that theresulting construct contains the cDNA in the correct orientation andsuch that the heterologous promoter can drive expression of thetranscription unit. The resulting expression constructs are introducedinto appropriate cell lines including but not limited to COS-7 cells (anAfrican Green Monkey Kidney cell line), HEK 293 cells (a human embryonickidney cell line) and NIH3T3 cells (a murine fibroblastic cell line).The DNA is introduced via standard methods (Sambrook et al 1989Molecular Cloning Second Edition, Cold Spring Harbour Laboratory Press)including but not limited to calcium phosphate transfection,electroporation or lipofectamine (Gibco) transfection. After therequired incubation time at 37° C. in a humidified incubator the cellsare tested for the presence of an active rat DRG sodium channel usingmethods described in example 7.

ii/Stable Expression

The production of a stable expression system has several advantages overtransient expression. A clonal cell line can be generated that a has astable phenotype and in which the expression levels of the foreignprotein can be characterised and, with some expression systems,controlled. Also, a range of vectors are available which incorporategenes coding for antibiotic resistance, thus allowing the selection ofcells transfected with the constructs introduced. Cell lines of thistype can be grown in tissue culture and can be frozen down for long-termstorage. There are several systems available for accomplishing this e.g.CHO, CV-1, NIH-3T3.

Specifically COS-7 cells can be transfected by lipofection usingLipofectamine (GibcoBRL) in the following manner. For each sample 2×10⁶cells are seeded in a 90 mm tissue culture plate the day prior totransfection. These are incubated overnight at 37° C. in a CO₂ incubatorto give 50-80% confluency the following day. The day of the transfectionthe following solutions are prepared in sterile 12×75 mm tubes: SolutionA: For each transfection, dilute 10-50 μg of DNA into 990 μl ofserum-free media (Opti-MEM I Reduced Serum Medium GibcoBRL). Solution B:For each transfection, dilute 50 μl of Lipofectamine Reagent into 950 μlserum-free medium. The two solutions are combined, mixed gently andincubated at room temp for 45 minutes. During this time the cells arerinsed once with serum-free medium. For each transfection 9 ml ofserum-free medium is added to the DNA-lipofectamine tubes. This solutionis mixed gently and overlayed on the rinsed cells. The plates areincubated for 5 hours at 37° C. in a CO₂ incubator. After the incubationthe medium is replaced with fresh complete media and the cells returnedto the incubator. Cells are assayed for activity 72 hours posttransfection as detailed in examples 4 and 7. To ascertain theefficiency of transfection, β-galactosidase in pcDNA3 is transfectedalongside the DRG sodium channel cDNA. This control plate is stained for13-galactosidase activity using a chromogenic substrate and theproportion of cells staining calculated. For transient transfection ofDRG the cDNA must first be cloned into a eucaryotic expression vectorsuch as pcDNA3 (Invitrogen).

EXAMPLE 6

Expression of Rat DRG Sodium Channel in Insect Cells

The baculovirus expression system uses baculovirus such as Autographacalifornica nuclear polyhedrosis virus (AcNPV) to produce large amountsof target protein in insect cells such as the Sf9 or 21 clonal celllines derived from Spodoptera frugiperda cells. Expression of the highlyabundant polyhedrin gene is non-essential in tissue culture and itsstrong promoter (polh) can be used for the synthesis of foreign geneproducts (Smith et al 1983 Mol Cell Biol 3,2156-2165). The polyhedrinpromoter is maximally expressed very late in infection (20 hours postinfection).

A transfer vector, where the rat DRG sodium channel cDNA is cloneddownstream of the polh promoter, or another late promoter such as p10,is transfected into insect cells in conjunction with modified AcNPVviral DNA such as but not limited to BaculoGold DNA (PharMingen). Themodified DNA contains a lethal mutation and is incapable of producinginfectious viral particles after transfection. Co-transfection with acomplementing transfer vector such as (but not limited to) pAcYM1(Matsuura et al 1987 J Gen Virol 68,1233-1250) or pVL1392/3 (InVitrogen)allows the production of viable recombinant virus. Although more than99% of the resultant virus particles should be derived fromplasmid-rescued virus it is desirable to further purify the virusparticles by plaque assay. To ensure that the recombinant stock isclonal, a single plaque is picked from the plaque assay and amplified toproduce a recombinant viral stock. Once the recombinant phenotype isverified the viral stock can be used to infect insect cells and expressfunctional rat DRG sodium channel. There are a number of variations inthe methodology of baculovirus expression which may give increasedexpression (O'Reilly et al 1992 Baculovirus Expression Vectors: ALaboratory Manual. Oxford University Press). The expression of the rator human DRG sodium channel is achieved by cloning of the cDNA intopVL1392 and introducing this into Sf21 insect cells.

EXAMPLE 7

Electrophysiological Characterisation of Cloned Human and Rat DRG SodiumChannel Expression

Xenopus laevis oocytes are used to express the channel after injectionof the mRNA or cDNA in an expression vector. Expression would betransient and thus functional studies would be made at appropriate timesafter the injections. Comparison with mock-injected oocytes woulddemonstrate lack of the novel channel as an endogenously expressedcharacteristic. Standard two electrode voltage clamp (TEVC) techniquesas described, for example, in Fraser, Moon & Djamgoz (1993)Electrophysiology of Xenopus oocytes: an expression system in molecularneurobiology. In: Electrophysiology: A practical approach. Wallis, D.I., ed. Oxford University Press. Chapter 4 pp. 65-86, would be used toexamine the characteristics of responses of ionic currents to changes inthe applied membrane potential. Appropriately modified saline mediawould be used to manipulate the type of ionic currents detectable. Thekinetics of activation and inactivation of the sodium current, its ionicselectivity, the effects of changes in ionic concentration of theextracellular medium on its reversal potential, and the sensitivity (orresistance) to TTX would be defining characteristics.

Similar electrophysiological studies would be undertaken to assess thesuccess of functional expression in a permanently or transientlyexpressing mammalian cell line, but patch clamp methods would be moresuitable than TEVC. Whole cell, cell-attached patch, inside-out patch oroutside-out patch configurations as described for example by Hamill etal. (1981) Pflugers Arch. 391:85-100 and Fenwick et al. (1982) J.Physiol. 331 599-635 might be used to assess the channelcharacteristics.

For example, isolated transfected cells (see above) will bevoltage-clamped using the whole-cell variant of the patch clamptechnique for recording the expressed sodium channel current.

Recordings will be obtained at room temperature (22-24° C.). Bothexternal and internal recording solutions will be used to isolate Na+currents as previously described (Lalik et al., Am. J. Physiol.264:C803-C809, 1992; West et al., Neuron 8:59-70, 1992). Externalsolution (mM): sodium chloride, 65; choline chloride, 50; TEA-Cl, 20,KCl, 1.5; calcium chloride, 1; magnesium chloride, 5; glucose 5; HEPES,5; at a pH 7.4 and and osmolality of 320. Internal solution (mM):CsF,90; CsCl, 60; sodium chloride, 10; MgCl, 2;EGTA, 10; HEPES, 10 at pH 7.2and an osmolarity of 315.

The kinetics and voltage parameters of the expressed sodium channelcurrent will be examined and compared with data existing in theliterature. These include current-voltage relationships and peak currentamplitude. Cells will be voltage-clamped at −70 mV and depolarizingpulses to 50 mV (at 10 mV increments) will be used to generate currents.

The pharmacology of the expressed sodium channel current will beexamined with the Na channel blocker, tetrodotoxin (TTX). To date sodiumchannels have been classified as TTX-sensitive and TTX-resistant: blockby low (1-30 nM) and high (>1 μM) concentrations of TTX, respectively(Elliot & Elliot, J. Physiol. (Lond.) 463:39-56, 1993; Yang et al., J.Neurosci. 12:268-277, 1992; W1992).

The channel is unaffected by concentrations lower than 1 micromolartetrodotoxin, and is only partially blocked by concentrations as high as10 micromolar tetrodotoxin.

EXAMPLE 8

Production of Purified Channel

Using a commercial coupled transcription-translation system, 35-Smethionine labelled protein products of the SNS clone can be generated(see FIG. 3). The size of the resulting protein when assessed bySDS-polyacrylamide gel electrophoresis confirms the predicted size ofthe protein deduced by DNA sequencing. The system used is the PromegaTNT system (Promega Technical Bulletin 126 1993). The experiment iscarried out precisely according to the protocol provided (see FIG. 3).

EXAMPLE 9

Use of Rat or Human Sodium Channel in Screening Assays

Cell lines expressing the cloned sodium channels could be used todetermine the effects of drugs on the ability of the channels to passsodium ions across the cell membranes, e.g to block the channels or toenhance their opening. Since the channel activation is voltagedependent, depolarising conditions will be required for observation ofbaseline activity that would be modified by drug actions. Depolarisationcould be achieved by for example raising extracellular potassium ionconcentration to 20 or 40 mM, or by repeated electrical pulses.Detection of the activation of sodium conducting channels could beachieved by flux of radiolabelled sodium ions, guanidine or by reportergene activation leading to for example a colour change or tofluorescence of a light emitting protein. Subsequent confirmation of theeffectiveness of the drug action on sodium channel activity wouldrequire electrophysiological studies similar to those described above.

EXAMPLE 10

In vitro Influx Assays

1. 22Na+ influx assay: A modified assay has been adapted from methodsreported by Tamkum and Catterall, Mol Pharm. 19:78, (1981). Oocytes orcells expressing the sodium channel gene are suspended in a buffercontaining 0.13 M sodium chloride, 5 mM KCl, 0.8 mM MgSO₄, 50 mMHEPES-Tris (pH 7.4), and 5.5 mM glucose. Aliquots of the cell suspensionare added a buffer containing 22NaCl (1.3 μCi/ml, New England Nuclear,Boston, Mass.), 0.128 M choline chloride, 2.66 mM sodium chloride, 5.4mM KCl, 0.8 mM MgSO₄, 50 mM HEPES-Tris (pH 7.4), 5 mM ouabain, 1 mg/mlbovine serum albumin, and 5.5 mM glucose and then incubated at 37° C.for 20 sec in either the presence or absence of 100 μM veratridine(Sigma Chemical Co., St Louis, Mo.). The influx assay is stopped by theaddition of 3 ml of ice-cold wash buffer containing 0.163 M sodiumchloride, 0.8 mM MgSO₄, 1.8 mM CaCl₂, 50 mM HEPES-Tris (pH 7.4) and 1mg/ml bovine serum albumin, collected on a glass fiber filter (WhatmanGF/C), and washed twices with 3 ml of wash buffer. Radioactiveincorporation is determined by with a gammacounter. The specifictetrodotoxin-resistant influx is measured by the difference in 22Na+uptake in the absence or the presence of 10 μM transmethrin or 1 μM (+)trans allethrin. The tetrodotoxin-sensitive influx is measured by thedifference in 22Na+ uptake in the absence or the presence of 1 1μMtetrodotoxin (Sigma Chemical Co., St Louis, Mo.).

Guanidine influx: Another assay is modified from the method described byReith, Eur. J. Pharmacol. 188:33 (1990). In this assay sodium ions aresubstituted with guanidinium ions. Oocytes or cells are washed twicewith a buffer containing 4.74 mM KCl, 1.25 mM CaCl₂, 1.2 mM KH2PO4, 1.18mM MgSO₄, 22 mM HEPES (pH 7.2), 22 mM choline chloride and 11 mMglucose. The oocytes or cells are suspended in the same buffercontaining 250 μM guanidine for 5 min at 19-25° C. An aliquot of14C-labelled guanidine hydrochloride (30-50 mCi/mmol supplied by NewEngland Nuclear, Boston, Mass.) is added in the absence or presence of10 μM veratridine, and the mixture is incubated for 3 min. The uptakereaction is stopped by filtration through Whatman GF/F filters andfollowed by 25 ml washes with ice-cold 0.9% saline. Radioactiveincorporation is determined by scintillation counting.

EXAMPLE 11

In order to measure the expression of sodium channels in in vitrosystems, as well as to analyse distribution and relative level ofexpression in vivo, and to attempt to block function, polyclonal andmonoclonal antibodies will be generated to peptide and protein fragmentsderived from SNS protein sequence shown in FIG. 1.

a) Immunogens

Glutathione-sulphotransferase (GST)—fusion proteins will be constructed(Smith and Johnson Gene 67:31-40 (1988)) using PGEX vectors obtainedfrom Pharmacia. Fusion proteins including both intracellular andextracellular loops with little homology with known sodium channelsother than SNS-B will be produced. One such method involves subcloningof fragments into pGex-5X3 or pGEX 4t-2 to produce in-frame fusionproteins encoding extracellular, intracellular or C-terminal domains asshown in detailed maps in FIG. 4. The pGEX fusion vectors aretransformed into E. coli XL-1 blue cells or other appropriate cellsgrown in the presence of ampicillin. After the cultures have reached anoptical density of OD600>0.5, fusion protein synthesis is induced by theaddition of 100 micromolar IPTG, and the cultures further incubated for1-4 hours. The cells are harvested by centrifugation and washed in icecold phosphate buffered saline. The resulting pellet (dissolved in 300microliters PBS from each 50 ml culture) is then sonicated on ice usinga 2 mm diameter probe, and the lysed cells microfuged to remove debris.50 microliters of glutathione-agarose beads are then added to eachpellet, and after gentle mixing for 2 minutes at room temperature, thebeads are washed by successive spins in PBS. The washed beads are thenboiled in Laemmli gel sample buffer, and applied to 10% polyacrylamideSDS gels. Material migrating at the predicted molecular weight isidentified on the gel by brief staining with coommassie blue, andcomparison with molecular weight markers. This material is thenelectroeluted from the gel and used as an immunogen as described below.

b) Antibody Production

Female Balb/c mice are immunised intraperiteonally with 1-100 microgramsof GST fusion protein emulisfied in Freunds complete adjuvant. After 4weeks, the animals will be further immunised with fusion proteins (1-100micrograms) emulsified in Freunds incomplete adjuvant. Four weeks later,the animals will be immunised intraperitoneally with a further 1-100micrograms of GST fusion protein emulsified with Freunds incompleteadjuvant. Seven days later, the animals will be tail bled, and theirserum assessed for the production of antibodies to the immunogen by thefollowing screen; (protocols for the production of rabbit polyclonalserum are the same, except that all injections are subcutaneous, and 10times as much immunogen is used. Polyclonal rabbit serum are isolatedfrom ear-vein bleeds.)

Serial ten-fold dilutions of the sera (1;100 to 1; 1000,000) inphosphate buffered saline (PBS) containing 0.5% NP-40 and 1% normal goatserum will be applied to 4% paraformaldehyde-fixed 10 micron sections ofneonatal rat spinal cord previously treated with 10% goat serum in PBS,After overnight incubation, the sections are washed in PBS, and furtherincubated in the dark with 1;200 FITC-conjugated F(ab)2 fragment of goatanti-mouse antibodies for 2 hours in PBS containing 1% normal goatserum. The sections are further washed in PBS, mounted in Citifluor, andexamined by fluorescence microscopy. Those sera that show specificstaining of laminar II in the spinal cord will be retained, and the micegenerating such antibodies subsequently used for the production ofmonoclonal antibodies. Three weeks later, mice producing usefulantibodies are immunised with GST—fusion proteins without adjuvant.After 3 days, the animals are killed, their spleens removed, and thelymphocytes fused with the thymidine kinase-negative myeloma line NSO orequivalent, using polyethylene glycol. The fused cells from eachexperiment are grown up in 3×24 well plates in the presence of DMEMmedium containing 10% fotal calf serum and hypoxanthine, aminopterin andthymidine (HAT) medium to kill the myeloma cells (Kohler and Milstein,Eur. J. Immunol 6, 511-519 (1976)). The tissue culture supernatants fromwells containing hybridomas are further screened by immunofluorescenceas described above, and cells from positive wells cloned by limitingdilution. Antibody from the positive testing cloned hybridomas is thenused to Western blot extracts of rat dorsal root ganglia, to detemine ifthe antibody recognises a band of size approximately 200,000, confirmingthe specificity of the monoclonal antibody for the SNS sodium channel.Those antibodies directed against extracellular domains that testpositive by both of these criteria will then be assessed for functionblocking activity in electrophysiological tests of sodium channelfunction (see example 7), and in screens relying on ion flux ordye-based assays in cells lines expressing sodium channel (see examples9 and 10).

EXAMPLE 12

Cell-type Distribution of Expression

In situ hybridization demonstrates the presence of SNS in a subset ofsensory neurons. An SNS fragment between positions 1740 and 1960 wassub-cloned into pGem4z, and DIG-UTP labeled sense or antisense cRNAgenerated. Sample preparation, hybridization, and visualization of insitu hybridization with alkaline phosphatase conjugated anti-DIGantibodies was carried out exactly as described in Schaeren-Wimers N.and Gerfin-Moser A. Histochemistry 100, 431-440 (1993).

EXAMPLE 13

Electrophysiological Properties of the Rat DRG Sodium Channel Expressedin Xenopus Oocytes

pBluescript SK plasmid containing DNA encoding the SNS sodium channelwas digested to position-21 upstream of the initiator methionine using acommercially available kit (Erase a base system, Promega, Madison, Wis.,USA). The linearized and digested plasmid was cut with Kpn 1 andsubcloned into an oocyte expression vector pSp64GL (Sma-Kpn1) sites.pSP64GL is derived from pSP64.T pSP64.T was cut with Sma1-EcoRI,blunt-ended with Klenow enzyme, and recircularized. Part of the pGem 72(+) polylinker (Sma1-Kpn1-EcoR1-Xhol) was ligated into the blunt-endedBg1 II site of pSP64.T. This vector with an altered polylinker for DNAinserts (Sma1-Kpn1-EcoR1-Xhol) and linearization (Sal1-Xba 1-BamH1) wasnamed pSP64GL. The resulting plasmid was linearized with Xba1, and cRNAtranscribed with SP6 polymerase using 1 mM 7-methylGppG.

cRNA (70 ng) was injected into Xenopus oocytes 7-14 days beforerecording; immature, stage IV oocytes were chosen cause of their smallerdiameter and therefore capacitance. Oocytes were impaled with 3M KClelectrodes (<1 MΩ) and perfused at 3-4 ml per minute with modifiedRinger solution containing 115 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1.8 mMMgCl₂, and 1 mM CaCl₂, pH 7.2, at temperature of 19.5-20.5° C. Digitalleak substraction of two electrode voltage-clamp current records wascarried out using as leak currents produced by hyperpolarizing pulses ofthe same amplitude as the test depolarizing commands. Oocytes in whichleak commands elicited time-dependent currents were discarded. Averagesof 10 records were used for both test and leak.

Inward currents were evoked by depolarizing, in 10 mV steps, from −60 mVto a command potential of −20 to +40 mV in 10 mV steps and from −80 mVto a command potential of −30 to +2− mV in oocytes injected with sodiumchannel cRNA. Current traces are blanked for the first 1.5 ms from theonset of the voltage step to delete the capacity transients for clarity.The peak current is reached at the same command voltage for the twoholding potentials, but is slightly smaller from −60 mV because ofsteady-state inactivation.

The effects of 50% or 100% replacement of external Na+ byN-methyl-D-glucosamine on the sodium channel current were elicited bystepping the depolarizing currents given to the oocyte from −60 to +1mV. Data were fitted with the equation h_(x)=1/(1+exp((V−V₅₀)/k)), whereV is the prepulse potential, V₅₀ the potential of 50% inactivation andthe k the slope factor (best squares fit). The effect of TTX (10 μM and100 μM) on the peak Na⁺ current (test pulse from −60 to +20 mV) was alsodetermined. The effect was quickly reversible upon washout.

After a minimum incubation of 7 days from cRNA injection, stepdepolarizations to potentials positive to −30 mV elicited inwardcurrents which peaked between +10 and +20 mV with an average maximumamplitude of 164±72 nA (from −60 mV holding potential, n=13) and areversal potential of +35.5±2.2 mV (n=10). The inward current wasreversed by total replacement of Na+ in the external medium with animpermeant cation (N-methyl-D-glucosamine). The current's reversalpotential was shifted in 50% Na+ by 13.7±3.2 mV in the hyperpolarizingdirection (n=3; predicted value for a Na+-selective channel, 17.5 mV).The inactivation produced by a 1s prepulse was half-maximal at -30.0±1.3mV (slope factor 14.0±1.7 mV, n=5.

TTX had no effect at nanomolar concentrations, and produced only a19.1±8.3% reduction at 10 μM, n=3). The estimated half-maximalinhibitory concentration (IC₅₀) was 59.6±10.1 μM TTX.

The local anesthetic lignocaine was also weakly inhibitory, producing amaximum block of 41.7±5.4% at 1 mM on the peak current elicited bydepolarizing pulses from −60 mV to +10 mV (1 every min; n=3), whereasunder the same conditions 100 μM phenytoin had no effect.

A similarity with the TTX-insensitive Na+ current of DRG neurons was theeffectiveness and rank order of Pb²⁺ versus Cd²⁺ in reducing peak Na⁺currents (−63.9±18.1% for Pb²⁺ versus −24.4±7.9% for Cd²⁺ at 50 μM and100 μM, respectively; n=3, P=0.0189). The electrophysiological andpharmacological characteristics of the oocyte expressed DRG sodiumchannel are thus similar to the properties of the sensory neuronTTX-insensitive channel, given the constraints of expression in anoocyte system. In oocytes expressing the DRG sodium channel, the peak ofthe I/V plot occurred at a more depolarized potential than that of theDRG TTX-insensitive current, despite a similar reversal potential. Thisdifference may reflect the absence of the accessory β1 subunit found inDRG, which is known to shift activation to more negative potentials whenexpressed with the subunit of other Na⁺ channels. In addition, splicevariants that exhibit an activation threshold more negative to SNSsodium channel may shift activation to the more negative potentialsobserved in sensory neurons.

EXAMPLE 14

Distribution of DRG Sodium Channel in Neonatal and Adult Rat TissuesandCell Lines

Northern blot and reverse transcriptase-polymerase chain reaction(RT-PCR) were used to examine neonatal and adult rat tissues forexpression of the DRG sodium channel messenger RNA.

Random primed ³²P-labeled DNA Pst -Acc1 fragment probes (50 ng, specificactivity 2×10⁹c.p.m. per μg DNA) from interdomain region 1 (nucleotideposition 1,478-1,892) of the SNS sodium channel nucleic acid sequencewere used to probe total RNA extracted from tissues. The followingtissues and cell lines were tested: central nervous system andnon-neuronal tissues from neonatal rats; peripheral nervous tissueincluding neonatal Schwann cells and sympathetic neurons, as well as C6glioma, human embryonal carcinoma line N-tera-2 and N-tera-2 neuro, ratsensory neuron-derived lines ND7 and ND8, and human neuroblastomasSMS-KCN and PC12 cells grown in the presence of NGF; adult rat tissueincluding pituitary, superior cervical ganglia, coeliac ganglia,trigeminal mesencephalic nucleus, vas deferens, bladder, ileum and DRGof adult animals treated with capsaicin (50 mg/kg) at birth and neonatalDRG control. Total RNA (10 μg) or 25 μg of RNA from tissues apart fromsuperior cervical ganglion sample (10 μg) and capsaicin-treated adultrat DRG (5 μg) were northern blotted.

Total RNA was separated on 1.2% agarose-formaldehyde gels, and capillaryblotted onto Hibond-N filters (Amersham). The amounts of RNA on the blotwere roughly equivalent, as judged by ethidium bromide staining ofribosomal RNA and by hybridization with the ubiquitously expressed L-27ribosomal protein transcripts. Filters were prehybridized in 50%formamide, 5×SSC containing 0.5% sodium dodecyl sulfate, 5×Denhardtssolution, 100 μg/ml boiled sonicated salmon sperm DNA (average size 300bp),10 μg/ml poly-U and 10 μg/ml poly-C at 45° C. for 6 h. After 36hours hybridization in the same conditions using 10⁷ c.p.m. per mlhybridization probe, the filters were briefly washed in 2×SSC at roomtemperature, then twice with 2×SSC with 0.5% SDS at 68° C. for 15 min,followed by a 20 min wash in 0.5% SDS, 0.2×SSC at 68° C. The filterswere autoradiographed overnight or for 4 days on autoradiography film(Kodak X-omat).

For RT-PCR experiments, 10 μg total RNA from neonatal rat tissues(spleen, liver, kidney, lung, intestine, muscle, heart, superiorcervical ganglia, spinal cord, brain stem, hippocampus, cerebellum,cortex and dorsal root ganglia), or 2 μg total RNA from control orcapsaicin-treated rat DRG or DRG neurons in culture were treated withDNase I and extracted with acidic phenol to remove genomic DNA.

cDNA was synthesized with Superscript reverse transcriptase using oligodT(12-18) primers and purified on Qiagen 5 tips. Polymerase chainreaction (PCR) was used to amplify cDNA (35 cycles, 94° C., 1 min; 55°C., 1 min; and 72° C., 1 min), and products separated on agarose gelsbefore staining with ethidium bromide. L-27 primers (Ninkina et al.(1983) Nucleic Acids Res. 21, 3175-3182) were added to the PCR reaction5 cycles after the start of the reaction with the DRG sodium channelspecific primers which comprised

5′-CAGCTTCGCTCAGAAGTATCT-3+ (SEQ ID NO: 9) and

5′-TTCTCGCCGTTCCACACGGAGA-3′ (SEQ ID NO: 10).

Transcription of mRNA coding for the DRG sodium channel could not bedetected in any non-neuronal tissues or in the central nervous systemusing northern blots or reverse transcription of mRNA and the polymerasechain reaction. Sympathetic neurons from the superior cervical ganglionand Schwann cell-containing sciatic nerve preparations, as well asseveral neuronal cell lines were also negative. However, total RNAextracts from neonatal and adult rat DRG gave a strong signal of sizeabout 7 kb on northern blots. These data suggest that the DRG sodiumchannel is not expressed only in early development.

RT-PCR of oligo dT-primed cDNA from various tissues using DRG sodiumchannel primers and L-27 ribosomal protein primer showed the presence ofDRG sodium channel transcripts in DRG tissue only.

RT-PCR was also performed on DRG-sodium channel and L-27 transcriptsfrom DRG neurons cultured and treated with capsaicin (overnight 10 μM)or dissected from neonatal animals treated with capsaicin (50 mg/kg on 2consecutive days, followed by DRG isolation 5 days later. The signalfrom the L-27 probe was the same in capsaicin-treated cell cultures oranimals as compared with controls that were not treated with capsaicin.There was a significant diminution in the DRG sodium channel signal fromcapsaicin-treated cultures or animals as compared with controls. ControlPCR reactions without reverse transcriptase treatment were also done tocontrol for contaminating genonic DNA.

When neonatal rats were treated with capsaicin and total adult DRG RNAsubsequently examined by northern blotting, the signal was substantiallyreduced, suggesting that the DRG sodium channel transcript is expressedselectively by capsaicin-sensitive (predominantly nociceptive) neurons.These data were confirmed by RT-PCF experiments on both cultures of DRGneurons, and in whole animal studies.

EXAMPLE 15

Distribution of DRG Sodium Channel in Rat Tissue by in situHybridization

In situ hybridization was used to examine the expression of the DRGsodium channel transcripts at the single-cell level in both adulttrigeninal ganglia and neonatal and adult rat DRG.

A SNS sodium channel PCR fragment of interdomain region I betweenpositions 1,736 and 1,797 of the SNS sodium channel nucleic acidsequence was subcloned into pGem3Z (Promega, Madison, Wis., USA) anddigoxygenin (DIG)-UTP (Boehringer-Mannheim, Germany) labeled sense orantisense cRNA generated using SP6 or T7 polymerase, respectively.Sample preparation, hybridization and visualization of in situhybridization with alkaline phosphatase conjugated anti-DIG antibodieswas carried out as described in Schaeren-Wimers, et al., A. (1993)Histochemistry 100: 431-440, with the following modifications. Frozentissue sections (10 μM-thick) of neonatal rat lumbar DRG, and adulttrigeminal ganglion neurons were fixed for 10 min in phosphate bufferedsaline (PBS) containing 4% paraformaldehyde. Sections were acetylated in0.1 M triethanolamine, 0.25% acetic anhydride for 10 min.Prehybridization was carried out in 50% formamide, 4×SSC, 100 μg/mlboiled and sonicated ssDNA, 50 μg/ml yeast tRNA, 2×Denhardts solution atroom temperature for 1 h. Hybridization was carried out overnight in thesame buffer at 65° C. Probe concentration was 50 ng/ml. Sections werewashed in 2×SSC for 30 min at 72° C. for 1 hr and twice in 0.1 SSC for30 min at 72° C. before visualization at room temperature withanti-digoxygenin alkaline phosphatase conjugated antibodies. The samesections were then stained with mouse monoclonal antibody RT97 which isspecific for neurofilaments found in large diameter neurons.

Subsets of sensory neurons from both tissues showed intense signals witha DRG sodium channel-specific probe. Combined immunohistochemistry withthe large-diameter neuron-specific monoclonal antibody RT97 and the DRGsodium channel specific probe showed that most of the large diameterneurons did not express the DRG sodium channel transcript. Smalldiameter neurons were stained with the DRG sodium channel specific probebut not the large diameter neurons.

EXAMPLE 16

Site Directed Mutagenesis of SNS Sodium Channel-TTX Sensitivity

The SNS sodium channel is 65% homologous to the tetrodotoxin-insensitivecardiac sodium channel. A number of residues that line the channelatrium have been implicated in tetrodotoxin binding. The amino acidsequence of the SNS sodium channel exhibits sequence identity to othertetrodotoxin-sensitive sodium channels in 7 out of 9 such residues. Onedifference is a conservative substitution at D(905)E. A single residue(C-357) has been shown to play a critical role in tetrodotoxin bindingto the sodium channel. In the SNS sodium channel, a hydrophilic serineis found at this position, whereasa other sodium channels that aresensitive to TTX have phenylalanine in this position.

Site-directed mutagenesis using standard techniques and primers havingthe sequence TGACGCAGGACTCCTGGGAGCGCC (SEQ ID NO: 31) was used tosubstitute phenylalanine for serine at position 357 in the SNS sodiumchannel. The mutated SNS sodium channel, when expressed in Xenopusoocytes produces voltage-gated currents similar in amplitude and timecourse to the native channel. However, sensitivity to TTX is restored togive an IC₅₀ of 2.5 nM (+−0.4, n=5), similar to other voltage-gatedsodium channels that have aromatic residues at the equivalant position.The table below shows IC₅₀ for SNS sodium channel, and the rat brainiia, muscle type 1, and cardiac tetrodotoxin-insensitive sodiumchannels.

TTX Sensitivity Sodium Channel ss1 domain ss2 domain IC₅₀ Rat brain iiaFRLM TQDFWENLY   18 nM muscle type 1 FRLM TQDYWENLY   40 nM cardiac TTXiFRLM TQDCWERLY  950 nM SNS FRLM TQDSWERLY   60 micromolar SNS mutantFRLM TQDFWERLY  2.5 nM

FRLM—SEQ ID NO: 11; TQDFWENLY—SEQ ID NO: 12;

TQDYWENLY—SEQ ID NO: 13; TQDCWERLY—SEQ ID NO: 14;

TQDSWERLY—SEQ ID NO: 15; TQDFWERLY—SEQ ID NO: 16

EXAMPLE 18

Polyclonal antibodies were raised in rabbits against the followingpeptides derived from the SNS sodium channel protein amino acidsequence:

Peptide 1 TQDSWER (SEQ ID NO:17)

Peptide 2 GSTDDNRSPQSDPYN (SEQ ID NO: 18)

Peptide 3 SPKENHGDFI (SEQ ID NO: 19)

Peptide 4 PNHNGSRGN (SEQ ID NO: 20)

The peptides were conjugated to Keyhole limpet heocyanin (KLH) andinjected repeatedly into rabbits. Sera from the rabbits was treated byWestern blotting. Several sera showed positive results indicating thepresence of antibodies specific for the peptide in the sera.

REFERENCES

Catterall W. A. (1992) Physiol. Rev. 72, S4-S47.

Cohen S. A. and Barchi R. L. (1993) Int. Rev. Cytology 137c, 55-103.

Hodgkin A. L. and Huxley A. F. (1952) J. Physiol. 116, 473-496.

Hille B. (1991) Ionic channels in excitable membranes (SinauerSunderland Mass.)

Jeftjina S. (1994) Brain Res. 639, 125-134.

Kohler G. and Milstein C. (1976) Eur J. Immunol 6, 511-519

Lewin B. (1995) Genes V Oxford University Press, Oxford.

Melton D. et al. (1984) Nucleic Acids Res. 12, 7035

Nowycky M. (1993) in Sensory Neurons (Ed Scott S.) OUP, Oxford.

Omri G. and Meir H. (1990) J. Membrane Biol. 115, 13-29

Pearce R. J. and Duchen M. R. (1994) Neuroscience 63, 1041-1056

Pfaff S L; Tamkun-M M; Taylor-W L (1990 Anal-Biochem. 1990 188 192-195

Schaeren-Wimers N. and Gerfin-Moser A. (1993) Histochemistry 100,431-440.

Smith D. B. and Johnson K. S. (1988) Gene 67, 31-40.

31 6524 base pairs nucleic acid single linear cDNA CDS 204..6077 1TAGCTTGCTT CTGCTAATGC TACCCCAGGC CTTTAGACAG AGAACAGATG GCAGATGGAG 60TTTCTTATTG CCATGCGCAA ACGCTGAGCC CACCTCATGA TCCCGGACCC CATGGTTTTC 120AGTAGACAAC CTGGGCTAAG AAGAGATCTC CGACCTTATA GAGCAGCAAA GAGTGTAAAT 180TCTTCCCCAA GAAGAATGAG AAG ATG GAG CTC CCC TTT GCG TCC GTG GGA 230 MetGlu Leu Pro Phe Ala Ser Val Gly 1 5 ACT ACC AAT TTC AGA CGG TTC ACT CCAGAG TCA CTG GCA GAG ATC GAG 278 Thr Thr Asn Phe Arg Arg Phe Thr Pro GluSer Leu Ala Glu Ile Glu 10 15 20 25 AAG CAG ATT GCT GCT CAC CGC GCA GCCAAG AAG GCC AGA ACC AAG CAC 326 Lys Gln Ile Ala Ala His Arg Ala Ala LysLys Ala Arg Thr Lys His 30 35 40 AGA GGA CAG GAG GAC AAG GGC GAG AAG CCCAGG CCT CAG CTG GAC TTG 374 Arg Gly Gln Glu Asp Lys Gly Glu Lys Pro ArgPro Gln Leu Asp Leu 45 50 55 AAA GAC TGT AAC CAG CTG CCC AAG TTC TAT GGTGAG CTC CCA GCA GAA 422 Lys Asp Cys Asn Gln Leu Pro Lys Phe Tyr Gly GluLeu Pro Ala Glu 60 65 70 CTG GTC GGG GAG CCC CTG GAG GAC CTA GAC CCT TTCTAC AGC ACA CAC 470 Leu Val Gly Glu Pro Leu Glu Asp Leu Asp Pro Phe TyrSer Thr His 75 80 85 CGG ACA TTC ATG GTG TTG AAT AAA AGC AGG ACC ATT TCCAGA TTC AGT 518 Arg Thr Phe Met Val Leu Asn Lys Ser Arg Thr Ile Ser ArgPhe Ser 90 95 100 105 GCC ACT TGG GCC CTG TGG CTC TTC AGT CCC TTC AACCTG ATC AGA AGA 566 Ala Thr Trp Ala Leu Trp Leu Phe Ser Pro Phe Asn LeuIle Arg Arg 110 115 120 ACA GCC ATC AAA GTG TCT GTC CAT TCC TGG TTC TCCATA TTC ATC ACC 614 Thr Ala Ile Lys Val Ser Val His Ser Trp Phe Ser IlePhe Ile Thr 125 130 135 ATC ACT ATT TTG GTC AAC TGC GTG TGC ATG ACC CGAACT GAT CTT CCA 662 Ile Thr Ile Leu Val Asn Cys Val Cys Met Thr Arg ThrAsp Leu Pro 140 145 150 GAG AAA GTC GAG TAC GTC TTC ACT GTC ATT TAC ACCTTC GAG GCT CTG 710 Glu Lys Val Glu Tyr Val Phe Thr Val Ile Tyr Thr PheGlu Ala Leu 155 160 165 ATT AAG ATA CTG GCA AGA GGG TTT TGT CTA AAT GAGTTC ACT TAT CTT 758 Ile Lys Ile Leu Ala Arg Gly Phe Cys Leu Asn Glu PheThr Tyr Leu 170 175 180 185 CGA GAT CCG TGG AAC TGG CTG GAC TTC AGT GTCATT ACC TTG GCG TAT 806 Arg Asp Pro Trp Asn Trp Leu Asp Phe Ser Val IleThr Leu Ala Tyr 190 195 200 GTG GGT GCA GCG ATA GAC CTC CGA GGA ATC TCAGGC CTG CGG ACA TTC 854 Val Gly Ala Ala Ile Asp Leu Arg Gly Ile Ser GlyLeu Arg Thr Phe 205 210 215 CGA GTT CTC AGA GCC CTG AAA ACT GTT TCT GTGATC CCA GGA CTG AAG 902 Arg Val Leu Arg Ala Leu Lys Thr Val Ser Val IlePro Gly Leu Lys 220 225 230 GTC ATC GTG GGA GCC CTG ATC CAC TCA GTG AGGAAG CTG GCC GAC GTG 950 Val Ile Val Gly Ala Leu Ile His Ser Val Arg LysLeu Ala Asp Val 235 240 245 ACT ATC CTC ACA GTC TTC TGC CTG AGC GTC TTCGCC TTG GTG GGC CTG 998 Thr Ile Leu Thr Val Phe Cys Leu Ser Val Phe AlaLeu Val Gly Leu 250 255 260 265 CAG CTC TTT AAG GGG AAC CTT AAG AAC AAATGC ATC AGG AAC GGA ACA 1046 Gln Leu Phe Lys Gly Asn Leu Lys Asn Lys CysIle Arg Asn Gly Thr 270 275 280 GAT CCC CAC AAG GCT GAC AAC CTC TCA TCTGAA ATG GCA GAA TAC GTC 1094 Asp Pro His Lys Ala Asp Asn Leu Ser Ser GluMet Ala Glu Tyr Val 285 290 295 TCC ATC AAG CCT GGT ACT ACG GAT CCC TTACTG TGC GGC AAT GGG TCT 1142 Ser Ile Lys Pro Gly Thr Thr Asp Pro Leu LeuCys Gly Asn Gly Ser 300 305 310 GAT GCT GGT CAC TGC CCT GGA GGC TAT GTCTGC CTG AAA ACT CCT GAC 1190 Asp Ala Gly His Cys Pro Gly Gly Tyr Val CysLeu Lys Thr Pro Asp 315 320 325 AAC CCG GAT TTT AAC TAC ACC AGC TTT GATTCC TTT GCG TGG GCA TTC 1238 Asn Pro Asp Phe Asn Tyr Thr Ser Phe Asp SerPhe Ala Trp Ala Phe 330 335 340 345 CTC TCA CTG TTC CGC CTC ATG ACG CAGGAC TCC TGG GAG CGC CTG TAC 1286 Leu Ser Leu Phe Arg Leu Met Thr Gln AspSer Trp Glu Arg Leu Tyr 350 355 360 CAG CAG ACA CTC CGG GCT TCT GGG AAAATG TAC ATG GTC TTT TTC GTG 1334 Gln Gln Thr Leu Arg Ala Ser Gly Lys MetTyr Met Val Phe Phe Val 365 370 375 CTG GTT ATT TTC CTT GGA TCG TTC TACCTG GTC AAT TTG ATC TTG GCC 1382 Leu Val Ile Phe Leu Gly Ser Phe Tyr LeuVal Asn Leu Ile Leu Ala 380 385 390 GTG GTC ACC ATG GCG TAT GAA GAG CAGAGC CAG GCA ACA ATT GCA GAA 1430 Val Val Thr Met Ala Tyr Glu Glu Gln SerGln Ala Thr Ile Ala Glu 395 400 405 ATC GAA GCC AAG GAA AAA AAG TTC CAGGAA GCC CTT GAG GTG CTG CAG 1478 Ile Glu Ala Lys Glu Lys Lys Phe Gln GluAla Leu Glu Val Leu Gln 410 415 420 425 AAG GAA CAG GAG GTG CTG GCA GCCCTG GGG ATT GAC ACG ACC TCG CTC 1526 Lys Glu Gln Glu Val Leu Ala Ala LeuGly Ile Asp Thr Thr Ser Leu 430 435 440 CAG TCC CAC AGT GGA TCA CCC TTAGCC TCC AAA AAC GCC AAT GAG AGA 1574 Gln Ser His Ser Gly Ser Pro Leu AlaSer Lys Asn Ala Asn Glu Arg 445 450 455 AGA CCC AGG GTG AAA TCA AGG GTGTCA GAG GGC TCC ACG GAT GAC AAC 1622 Arg Pro Arg Val Lys Ser Arg Val SerGlu Gly Ser Thr Asp Asp Asn 460 465 470 AGG TCA CCC CAA TCT GAC CCT TACAAC CAG CGC AGG ATG TCT TTC CTA 1670 Arg Ser Pro Gln Ser Asp Pro Tyr AsnGln Arg Arg Met Ser Phe Leu 475 480 485 GGC CTG TCT TCA GGA AGA CGC AGGGCT AGC CAC GGC AGT GTG TTC CAC 1718 Gly Leu Ser Ser Gly Arg Arg Arg AlaSer His Gly Ser Val Phe His 490 495 500 505 TTC CGA GCG CCC AGC CAA GACATC TCA TTT CCT GAC GGG ATC ACC CCT 1766 Phe Arg Ala Pro Ser Gln Asp IleSer Phe Pro Asp Gly Ile Thr Pro 510 515 520 GAT GAT GGG GTC TTT CAC GGAGAC CAG GAA AGC CGT CGA GGT TCC ATA 1814 Asp Asp Gly Val Phe His Gly AspGln Glu Ser Arg Arg Gly Ser Ile 525 530 535 TTG CTG GGC AGG GGT GCT GGGCAG ACA GGT CCA CTC CCC AGG AGC CCA 1862 Leu Leu Gly Arg Gly Ala Gly GlnThr Gly Pro Leu Pro Arg Ser Pro 540 545 550 CTG CCT CAG TCC CCC AAC CCTGGC CGT AGA CAT GGA GAA GAG GGA CAG 1910 Leu Pro Gln Ser Pro Asn Pro GlyArg Arg His Gly Glu Glu Gly Gln 555 560 565 CTC GGA GTG CCC ACT GGT GAGCTT ACC GCT GGA GCG CCT GAA GGC CCG 1958 Leu Gly Val Pro Thr Gly Glu LeuThr Ala Gly Ala Pro Glu Gly Pro 570 575 580 585 GCA CTG CAC ACT ACA GGGCAG AAG AGC TTC CTG TCT GCG GGC TAC TTG 2006 Ala Leu His Thr Thr Gly GlnLys Ser Phe Leu Ser Ala Gly Tyr Leu 590 595 600 AAC GAA CCT TTC CGA GCACAG AGG GCC ATG AGC GTT GTC AGT ATC ATG 2054 Asn Glu Pro Phe Arg Ala GlnArg Ala Met Ser Val Val Ser Ile Met 605 610 615 ACT TCT GTC ATT GAG GAGCTT GAA GAG TCT AAG CTG AAG TGC CCA CCC 2102 Thr Ser Val Ile Glu Glu LeuGlu Glu Ser Lys Leu Lys Cys Pro Pro 620 625 630 TGC TTG ATC AGC TTC GCTCAG AAG TAT CTG ATC TGG GAG TGC TGC CCC 2150 Cys Leu Ile Ser Phe Ala GlnLys Tyr Leu Ile Trp Glu Cys Cys Pro 635 640 645 AAG TGG AGG AAG TTC AAGATG GCG CTG TTC GAG CTG GTG ACT GAC CCC 2198 Lys Trp Arg Lys Phe Lys MetAla Leu Phe Glu Leu Val Thr Asp Pro 650 655 660 665 TTC GCA GAG CTT ACCATC ACC CTC TGC ATC GTG GTG AAC ACC GTC TTC 2246 Phe Ala Glu Leu Thr IleThr Leu Cys Ile Val Val Asn Thr Val Phe 670 675 680 ATG GCC ATG GAG CACTAC CCC ATG ACC GAT GCC TTC GAT GCC ATG CTT 2294 Met Ala Met Glu His TyrPro Met Thr Asp Ala Phe Asp Ala Met Leu 685 690 695 CAA GCC GGC AAC ATTGTC TTC ACC GTG TTT TTC ACA ATG GAG ATG GCC 2342 Gln Ala Gly Asn Ile ValPhe Thr Val Phe Phe Thr Met Glu Met Ala 700 705 710 TTC AAG ATC ATT GCCTTC GAC CCC TAC TAT TAC TTC CAG AAG AAG TGG 2390 Phe Lys Ile Ile Ala PheAsp Pro Tyr Tyr Tyr Phe Gln Lys Lys Trp 715 720 725 AAT ATC TTC GAC TGTGTC ATC GTC ACC GTG AGC CTT CTG GAG CTG AGT 2438 Asn Ile Phe Asp Cys ValIle Val Thr Val Ser Leu Leu Glu Leu Ser 730 735 740 745 GCA TCC AAG AAGGGC AGC CTG TCT GTG CTC CGT ACC TTA CGC TTG CTG 2486 Ala Ser Lys Lys GlySer Leu Ser Val Leu Arg Thr Leu Arg Leu Leu 750 755 760 CGG GTC TTC AAGCTG GCC AAG TCC TGG CCC ACC CTG AAC ACC CTC ATC 2534 Arg Val Phe Lys LeuAla Lys Ser Trp Pro Thr Leu Asn Thr Leu Ile 765 770 775 AAG ATC ATC GGGAAC TCA GTG GGG GCC CTG GGC AAC CTG ACC TTT ATC 2582 Lys Ile Ile Gly AsnSer Val Gly Ala Leu Gly Asn Leu Thr Phe Ile 780 785 790 CTG GCC ATC ATCGTC TTC ATC TTC GCC CTG GTC GGA AAG CAG CTT CTC 2630 Leu Ala Ile Ile ValPhe Ile Phe Ala Leu Val Gly Lys Gln Leu Leu 795 800 805 TCA GAG GAC TACGGG TGC CGC AAG GAC GGC GTC TCC GTG TGG AAC GGC 2678 Ser Glu Asp Tyr GlyCys Arg Lys Asp Gly Val Ser Val Trp Asn Gly 810 815 820 825 GAG AAG CTCCGC TGG CAC ATG TGT GAC TTC TTC CAT TCC TTC CTG GTC 2726 Glu Lys Leu ArgTrp His Met Cys Asp Phe Phe His Ser Phe Leu Val 830 835 840 GTC TTC CGAATC CTC TGC GGG GAG TGG ATC GAG AAC ATG TGG GTC TGC 2774 Val Phe Arg IleLeu Cys Gly Glu Trp Ile Glu Asn Met Trp Val Cys 845 850 855 ATG GAG GTCAGC CAG AAA TCC ATC TGC CTC ATC CTC TTC TTG ACT GTG 2822 Met Glu Val SerGln Lys Ser Ile Cys Leu Ile Leu Phe Leu Thr Val 860 865 870 ATG GTG CTGGGC AAC CTA GTG GTG CTC AAC CTT TTC ATC GCT TTA CTG 2870 Met Val Leu GlyAsn Leu Val Val Leu Asn Leu Phe Ile Ala Leu Leu 875 880 885 CTG AAC TCCTTC AGC GCG GAC AAC CTC ACG GCT CCA GAG GAT GAC GGG 2918 Leu Asn Ser PheSer Ala Asp Asn Leu Thr Ala Pro Glu Asp Asp Gly 890 895 900 905 GAG GTGAAC AAC TTG CAG TTA GCA CTG GCC AGG ATC CAG GTA CTT GGC 2966 Glu Val AsnAsn Leu Gln Leu Ala Leu Ala Arg Ile Gln Val Leu Gly 910 915 920 CAT CGGGCC AGC AGG GCC AGC GCC AGT TAC ATC AGC AGC CAC TGC CGA 3014 His Arg AlaSer Arg Ala Ser Ala Ser Tyr Ile Ser Ser His Cys Arg 925 930 935 TTC CACTGG CCC AAG GTG GAG ACC CAG CTG GGC ATG AAG CCC CCA CTC 3062 Phe His TrpPro Lys Val Glu Thr Gln Leu Gly Met Lys Pro Pro Leu 940 945 950 ACC AGCTCA GAG GCC AAG AAC CAC ATT GCC ACT GAT GCT GTC AGT GCT 3110 Thr Ser SerGlu Ala Lys Asn His Ile Ala Thr Asp Ala Val Ser Ala 955 960 965 GCA GTGGGG AAC CTG ACA AAG CCA GCT CTC AGT AGC CCC AAG GAG AAC 3158 Ala Val GlyAsn Leu Thr Lys Pro Ala Leu Ser Ser Pro Lys Glu Asn 970 975 980 985 CACGGG GAC TTC ATC ACT GAT CCC AAC GTG TGG GTC TCT GTG CCC ATT 3206 His GlyAsp Phe Ile Thr Asp Pro Asn Val Trp Val Ser Val Pro Ile 990 995 1000 GCTGAG GGG GAA TCT GAC CTC GAC GAG CTC GAG GAA GAT ATG GAG CAG 3254 Ala GluGly Glu Ser Asp Leu Asp Glu Leu Glu Glu Asp Met Glu Gln 1005 1010 1015GCT TCG CAG AGC TCC TGG CAG GAA GAG GAC CCC AAG GGA CAG CAG GAG 3302 AlaSer Gln Ser Ser Trp Gln Glu Glu Asp Pro Lys Gly Gln Gln Glu 1020 10251030 CAG TTG CCA CAA GTC CAA AAG TGT GAA AAC CAC CAG GCA GCC AGA AGC3350 Gln Leu Pro Gln Val Gln Lys Cys Glu Asn His Gln Ala Ala Arg Ser1035 1040 1045 CCA GCC TCC ATG ATG TCC TCT GAG GAC CTG GCT CCA TAC CTGGGT GAG 3398 Pro Ala Ser Met Met Ser Ser Glu Asp Leu Ala Pro Tyr Leu GlyGlu 1050 1055 1060 1065 AGC TGG AAG AGG AAG GAT AGC CCT CAG GTC CCT GCCGAG GGA GTG GAT 3446 Ser Trp Lys Arg Lys Asp Ser Pro Gln Val Pro Ala GluGly Val Asp 1070 1075 1080 GAC ACG AGC TCC TCT GAG GGC AGC ACG GTG GACTGC CCG GAC CCA GAG 3494 Asp Thr Ser Ser Ser Glu Gly Ser Thr Val Asp CysPro Asp Pro Glu 1085 1090 1095 GAA ATC CTG AGG AAG ATC CCC GAG CTG GCACAT GAC CTG GAC GAG CCC 3542 Glu Ile Leu Arg Lys Ile Pro Glu Leu Ala HisAsp Leu Asp Glu Pro 1100 1105 1110 GAT GAC TGT TTC AGA GAA GGC TGC ACTCGC CGC TGT CCC TGC TGC AAC 3590 Asp Asp Cys Phe Arg Glu Gly Cys Thr ArgArg Cys Pro Cys Cys Asn 1115 1120 1125 GTG AAT ACT AGC AAG TCT CCT TGGGCC ACA GGC TGG CAG GTG CGC AAG 3638 Val Asn Thr Ser Lys Ser Pro Trp AlaThr Gly Trp Gln Val Arg Lys 1130 1135 1140 1145 ACC TGC TAC CGC ATC GTGGAG CAC AGC TGG TTT GAG AGT TTC ATC ATC 3686 Thr Cys Tyr Arg Ile Val GluHis Ser Trp Phe Glu Ser Phe Ile Ile 1150 1155 1160 TTC ATG ATC CTG CTCAGC AGT GGA GCG CTG GCC TTT GAG GAT AAC TAC 3734 Phe Met Ile Leu Leu SerSer Gly Ala Leu Ala Phe Glu Asp Asn Tyr 1165 1170 1175 CTG GAA GAG AAACCC CGA GTG AAG TCC GTG CTG GAG TAC ACT GAC CGA 3782 Leu Glu Glu Lys ProArg Val Lys Ser Val Leu Glu Tyr Thr Asp Arg 1180 1185 1190 GTG TTC ACCTTC ATC TTC GTC TTT GAG ATG CTG CTC AAG TGG GTA GCC 3830 Val Phe Thr PheIle Phe Val Phe Glu Met Leu Leu Lys Trp Val Ala 1195 1200 1205 TAT GGCTTC AAA AAG TAT TTC ACC AAT GCC TGG TGC TGG CTG GAC TTC 3878 Tyr Gly PheLys Lys Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe 1210 1215 1220 1225CTC ATT GTG AAC ATC TCC CTG ACA AGC CTC ATA GCG AAG ATC CTT GAG 3926 LeuIle Val Asn Ile Ser Leu Thr Ser Leu Ile Ala Lys Ile Leu Glu 1230 12351240 TAT TCC GAC GTG GCG TCC ATC AAA GCC CTT CGG ACT CTC CGT GCC CTC3974 Tyr Ser Asp Val Ala Ser Ile Lys Ala Leu Arg Thr Leu Arg Ala Leu1245 1250 1255 CGA CCG CTG CGG GCT CTG TCT CGA TTC GAA GGC ATG AGG GTAGTG GTG 4022 Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val ValVal 1260 1265 1270 GAT GCC CTC GTG GGC GCC ATC CCC TCC ATC ATG AAC GTCCTC CTC GTC 4070 Asp Ala Leu Val Gly Ala Ile Pro Ser Ile Met Asn Val LeuLeu Val 1275 1280 1285 TGC CTC ATC TTC TGG CTC ATC TTC AGC ATC ATG GGCGTG AAC CTC TTC 4118 Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly ValAsn Leu Phe 1290 1295 1300 1305 GCC GGG AAA TTT TCG AAG TGC GTC GAC ACCAGA AAT AAC CCA TTT TCC 4166 Ala Gly Lys Phe Ser Lys Cys Val Asp Thr ArgAsn Asn Pro Phe Ser 1310 1315 1320 AAC GTG AAT TCG ACG ATG GTG AAT AACAAG TCC GAG TGT CAC AAT CAA 4214 Asn Val Asn Ser Thr Met Val Asn Asn LysSer Glu Cys His Asn Gln 1325 1330 1335 AAC AGC ACC GGC CAC TTC TTC TGGGTC AAC GTC AAA GTC AAC TTC GAC 4262 Asn Ser Thr Gly His Phe Phe Trp ValAsn Val Lys Val Asn Phe Asp 1340 1345 1350 AAC GTC GCT ATG GGC TAC CTCGCA CTT CTT CAG GTG GCA ACC TTC AAA 4310 Asn Val Ala Met Gly Tyr Leu AlaLeu Leu Gln Val Ala Thr Phe Lys 1355 1360 1365 GGC TGG ATG GAC ATA ATGTAT GCA GCT GTT GAT TCC GGA GAG ATC AAC 4358 Gly Trp Met Asp Ile Met TyrAla Ala Val Asp Ser Gly Glu Ile Asn 1370 1375 1380 1385 AGT CAG CCT AACTGG GAG AAC AAC TTG TAC ATG TAC CTG TAC TTC GTC 4406 Ser Gln Pro Asn TrpGlu Asn Asn Leu Tyr Met Tyr Leu Tyr Phe Val 1390 1395 1400 GTT TTC ATCATT TTC GGT GGC TTC TTC ACG CTG AAT CTC TTT GTT GGG 4454 Val Phe Ile IlePhe Gly Gly Phe Phe Thr Leu Asn Leu Phe Val Gly 1405 1410 1415 GTC ATAATC GAC AAC TTC AAC CAA CAG AAA AAA AAG CTA GGA GGC CAG 4502 Val Ile IleAsp Asn Phe Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln 1420 1425 1430 GACATC TTC ATG ACA GAA GAG CAG AAG AAG TAC TAC AAT GCC ATG AAG 4550 Asp IlePhe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys 1435 1440 1445AAG CTG GGC TCC AAG AAA CCC CAG AAG CCC ATC CCA CGG CCC CTG AAT 4598 LysLeu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Leu Asn 1450 14551460 1465 AAG TAC CAA GGC TTC GTG TTT GAC ATC GTG ACC AGG CAA GCC TTTGAC 4646 Lys Tyr Gln Gly Phe Val Phe Asp Ile Val Thr Arg Gln Ala Phe Asp1470 1475 1480 ATC ATC ATC ATG GTT CTC ATC TGC CTC AAC ATG ATC ACC ATGATG GTG 4694 Ile Ile Ile Met Val Leu Ile Cys Leu Asn Met Ile Thr Met MetVal 1485 1490 1495 GAG ACC GAC GAG CAG GGC GAG GAG AAG ACG AAG GTT CTGGGC AGA ATC 4742 Glu Thr Asp Glu Gln Gly Glu Glu Lys Thr Lys Val Leu GlyArg Ile 1500 1505 1510 AAC CAG TTC TTT GTG GCC GTC TTC ACG GGC GAG TGTGTG ATG AAG ATG 4790 Asn Gln Phe Phe Val Ala Val Phe Thr Gly Glu Cys ValMet Lys Met 1515 1520 1525 TTC GCC CTG CGA CAG TAC TAC TTC ACC AAC GGCTGG AAC GTG TTC GAC 4838 Phe Ala Leu Arg Gln Tyr Tyr Phe Thr Asn Gly TrpAsn Val Phe Asp 1530 1535 1540 1545 TTC ATA GTG GTG ATC CTG TCC ATT GGGAGT CTG CTG TTT TCT GCA ATC 4886 Phe Ile Val Val Ile Leu Ser Ile Gly SerLeu Leu Phe Ser Ala Ile 1550 1555 1560 CTT AAG TCA CTG GAA AAC TAC TTCTCC CCG ACG CTC TTC CGG GTC ATC 4934 Leu Lys Ser Leu Glu Asn Tyr Phe SerPro Thr Leu Phe Arg Val Ile 1565 1570 1575 CGT CTG GCC AGG ATC GGC CGCATC CTC AGG CTG ATC CGA GCA GCC AAG 4982 Arg Leu Ala Arg Ile Gly Arg IleLeu Arg Leu Ile Arg Ala Ala Lys 1580 1585 1590 GGG ATT CGC ACG CTG CTCTTC GCC CTC ATG ATG TCC CTG CCC GCC CTC 5030 Gly Ile Arg Thr Leu Leu PheAla Leu Met Met Ser Leu Pro Ala Leu 1595 1600 1605 TTC AAC ATC GGC CTCCTC CTC TTC CTC GTC ATG TTC ATC TAC TCC ATC 5078 Phe Asn Ile Gly Leu LeuLeu Phe Leu Val Met Phe Ile Tyr Ser Ile 1610 1615 1620 1625 TTC GGC ATGGCC AGC TTC GCT AAC GTC GTG GAC GAG GCC GGC ATC GAC 5126 Phe Gly Met AlaSer Phe Ala Asn Val Val Asp Glu Ala Gly Ile Asp 1630 1635 1640 GAC ATGTTC AAC TTC AAG ACC TTT GGC AAC AGC ATG CTG TGC CTG TTC 5174 Asp Met PheAsn Phe Lys Thr Phe Gly Asn Ser Met Leu Cys Leu Phe 1645 1650 1655 CAGATC ACC ACC TCG GCC GGC TGG GAC GGC CTC CTC AGC CCC ATC CTC 5222 Gln IleThr Thr Ser Ala Gly Trp Asp Gly Leu Leu Ser Pro Ile Leu 1660 1665 1670AAC ACG GGG CCT CCC TAC TGC GAC CCC AAC CTG CCC AAC AGC AAC GGC 5270 AsnThr Gly Pro Pro Tyr Cys Asp Pro Asn Leu Pro Asn Ser Asn Gly 1675 16801685 TCC CGG GGG AAC TGC GGG AGC CCG GCG GTG GGC ATC ATC TTC TTC ACC5318 Ser Arg Gly Asn Cys Gly Ser Pro Ala Val Gly Ile Ile Phe Phe Thr1690 1695 1700 1705 ACC TAC ATC ATC ATC TCC TTC CTC ATC GTG GTC AAC ATGTAC ATC GCA 5366 Thr Tyr Ile Ile Ile Ser Phe Leu Ile Val Val Asn Met TyrIle Ala 1710 1715 1720 GTG ATT CTG GAG AAC TTC AAC GTA GCC ACC GAG GAGAGC ACG GAG CCC 5414 Val Ile Leu Glu Asn Phe Asn Val Ala Thr Glu Glu SerThr Glu Pro 1725 1730 1735 CTG AGC GAG GAC GAC TTC GAC ATG TTC TAT GAGACC TGG GAG AAG TTC 5462 Leu Ser Glu Asp Asp Phe Asp Met Phe Tyr Glu ThrTrp Glu Lys Phe 1740 1745 1750 GAC CCG GAG GCC ACC CAG TTC ATT GCC TTTTCT GCC CTC TCA GAC TTC 5510 Asp Pro Glu Ala Thr Gln Phe Ile Ala Phe SerAla Leu Ser Asp Phe 1755 1760 1765 GCG GAC ACG CTC TCC GGC CCT CTT AGAATC CCC AAA CCC AAC CAG AAT 5558 Ala Asp Thr Leu Ser Gly Pro Leu Arg IlePro Lys Pro Asn Gln Asn 1770 1775 1780 1785 ATA TTA ATC CAG ATG GAC CTGCCG TTG GTC CCC GGG GAT AAG ATC CAC 5606 Ile Leu Ile Gln Met Asp Leu ProLeu Val Pro Gly Asp Lys Ile His 1790 1795 1800 TGT CTG GAC ATC CTT TTTGCC TTC ACA AAG AAC GTC TTG GGA GAA TCC 5654 Cys Leu Asp Ile Leu Phe AlaPhe Thr Lys Asn Val Leu Gly Glu Ser 1805 1810 1815 GGG GAG TTG GAC TCCCTG AAG ACC AAT ATG GAA GAG AAG TTT ATG GCG 5702 Gly Glu Leu Asp Ser LeuLys Thr Asn Met Glu Glu Lys Phe Met Ala 1820 1825 1830 ACC AAT CTC TCCAAA GCA TCC TAT GAA CCA ATA GCC ACC ACC CTC CGG 5750 Thr Asn Leu Ser LysAla Ser Tyr Glu Pro Ile Ala Thr Thr Leu Arg 1835 1840 1845 TGG AAG CAGGAA GAC CTC TCA GCC ACA GTC ATT CAA AAG GCC TAC CGG 5798 Trp Lys Gln GluAsp Leu Ser Ala Thr Val Ile Gln Lys Ala Tyr Arg 1850 1855 1860 1865 AGCTAC ATG CTG CAC CGC TCC TTG ACA CTC TCC AAC ACC CTG CAT GTG 5846 Ser TyrMet Leu His Arg Ser Leu Thr Leu Ser Asn Thr Leu His Val 1870 1875 1880CCC AGG GCT GAG GAG GAT GGC GTG TCA CTT CCC GGG GAA GGC TAC ATT 5894 ProArg Ala Glu Glu Asp Gly Val Ser Leu Pro Gly Glu Gly Tyr Ile 1885 18901895 ACA TTC ATG GCA AAC AGT GGA CTC CCG GAC AAA TCA GAA ACT GCC TCT5942 Thr Phe Met Ala Asn Ser Gly Leu Pro Asp Lys Ser Glu Thr Ala Ser1900 1905 1910 GCT ACG TCT TTC CCG CCA TCC TAT GAC AGT GTC ACC AGG GGCCTG AGT 5990 Ala Thr Ser Phe Pro Pro Ser Tyr Asp Ser Val Thr Arg Gly LeuSer 1915 1920 1925 GAC CGG GCC AAC ATT AAC CCA TCT AGC TCA ATG CAA AATGAA GAT GAG 6038 Asp Arg Ala Asn Ile Asn Pro Ser Ser Ser Met Gln Asn GluAsp Glu 1930 1935 1940 1945 GTC GCT GCT AAG GAA GGA AAC AGC CCT GGA CCTCAG TGAAGGCACT 6084 Val Ala Ala Lys Glu Gly Asn Ser Pro Gly Pro Gln 19501955 CAGGCATGCA CAGGGCAGGT TCCAATGTCT TTCTCTGCTG TACTAACTCC TTCCCTCTGG6144 AGGTGGCACC AACCTCCAGC CTCCACCAAT GCATGTCACT GGTCATGGTG TCAGAACTGA6204 ATGGGGACAT CCTTGAGAAA GCCCCCACCC CAATAGGAAT CAAAAGCCAA GGATACTCCT6264 CCATTCTGAC GTCCCTTCCG AGTTCCCAGA AGATGTCATT GCTCCCTTCT GTTTGTGACC6324 AGAGACGTGA TTCACCAACT TCTCGGAGCC AGAGACACAT AGCAAAGACT TTTCTGCTGG6384 TGTCGGGCAG TCTTAGAGAA GTCACGTAGG GGTTGGTACT GAGAATTAGG GTTTGCATGA6444 CTGCATGCTC ACAGCTGCCG GACAATACCT GTGAGTCGGC CATTAAAATT AATATTTTTA6504 AAGTTAAAAA AAAAAAAAAA 6524 1957 amino acids amino acid linearprotein 2 Met Glu Leu Pro Phe Ala Ser Val Gly Thr Thr Asn Phe Arg ArgPhe 1 5 10 15 Thr Pro Glu Ser Leu Ala Glu Ile Glu Lys Gln Ile Ala AlaHis Arg 20 25 30 Ala Ala Lys Lys Ala Arg Thr Lys His Arg Gly Gln Glu AspLys Gly 35 40 45 Glu Lys Pro Arg Pro Gln Leu Asp Leu Lys Asp Cys Asn GlnLeu Pro 50 55 60 Lys Phe Tyr Gly Glu Leu Pro Ala Glu Leu Val Gly Glu ProLeu Glu 65 70 75 80 Asp Leu Asp Pro Phe Tyr Ser Thr His Arg Thr Phe MetVal Leu Asn 85 90 95 Lys Ser Arg Thr Ile Ser Arg Phe Ser Ala Thr Trp AlaLeu Trp Leu 100 105 110 Phe Ser Pro Phe Asn Leu Ile Arg Arg Thr Ala IleLys Val Ser Val 115 120 125 His Ser Trp Phe Ser Ile Phe Ile Thr Ile ThrIle Leu Val Asn Cys 130 135 140 Val Cys Met Thr Arg Thr Asp Leu Pro GluLys Val Glu Tyr Val Phe 145 150 155 160 Thr Val Ile Tyr Thr Phe Glu AlaLeu Ile Lys Ile Leu Ala Arg Gly 165 170 175 Phe Cys Leu Asn Glu Phe ThrTyr Leu Arg Asp Pro Trp Asn Trp Leu 180 185 190 Asp Phe Ser Val Ile ThrLeu Ala Tyr Val Gly Ala Ala Ile Asp Leu 195 200 205 Arg Gly Ile Ser GlyLeu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys 210 215 220 Thr Val Ser ValIle Pro Gly Leu Lys Val Ile Val Gly Ala Leu Ile 225 230 235 240 His SerVal Arg Lys Leu Ala Asp Val Thr Ile Leu Thr Val Phe Cys 245 250 255 LeuSer Val Phe Ala Leu Val Gly Leu Gln Leu Phe Lys Gly Asn Leu 260 265 270Lys Asn Lys Cys Ile Arg Asn Gly Thr Asp Pro His Lys Ala Asp Asn 275 280285 Leu Ser Ser Glu Met Ala Glu Tyr Val Ser Ile Lys Pro Gly Thr Thr 290295 300 Asp Pro Leu Leu Cys Gly Asn Gly Ser Asp Ala Gly His Cys Pro Gly305 310 315 320 Gly Tyr Val Cys Leu Lys Thr Pro Asp Asn Pro Asp Phe AsnTyr Thr 325 330 335 Ser Phe Asp Ser Phe Ala Trp Ala Phe Leu Ser Leu PheArg Leu Met 340 345 350 Thr Gln Asp Ser Trp Glu Arg Leu Tyr Gln Gln ThrLeu Arg Ala Ser 355 360 365 Gly Lys Met Tyr Met Val Phe Phe Val Leu ValIle Phe Leu Gly Ser 370 375 380 Phe Tyr Leu Val Asn Leu Ile Leu Ala ValVal Thr Met Ala Tyr Glu 385 390 395 400 Glu Gln Ser Gln Ala Thr Ile AlaGlu Ile Glu Ala Lys Glu Lys Lys 405 410 415 Phe Gln Glu Ala Leu Glu ValLeu Gln Lys Glu Gln Glu Val Leu Ala 420 425 430 Ala Leu Gly Ile Asp ThrThr Ser Leu Gln Ser His Ser Gly Ser Pro 435 440 445 Leu Ala Ser Lys AsnAla Asn Glu Arg Arg Pro Arg Val Lys Ser Arg 450 455 460 Val Ser Glu GlySer Thr Asp Asp Asn Arg Ser Pro Gln Ser Asp Pro 465 470 475 480 Tyr AsnGln Arg Arg Met Ser Phe Leu Gly Leu Ser Ser Gly Arg Arg 485 490 495 ArgAla Ser His Gly Ser Val Phe His Phe Arg Ala Pro Ser Gln Asp 500 505 510Ile Ser Phe Pro Asp Gly Ile Thr Pro Asp Asp Gly Val Phe His Gly 515 520525 Asp Gln Glu Ser Arg Arg Gly Ser Ile Leu Leu Gly Arg Gly Ala Gly 530535 540 Gln Thr Gly Pro Leu Pro Arg Ser Pro Leu Pro Gln Ser Pro Asn Pro545 550 555 560 Gly Arg Arg His Gly Glu Glu Gly Gln Leu Gly Val Pro ThrGly Glu 565 570 575 Leu Thr Ala Gly Ala Pro Glu Gly Pro Ala Leu His ThrThr Gly Gln 580 585 590 Lys Ser Phe Leu Ser Ala Gly Tyr Leu Asn Glu ProPhe Arg Ala Gln 595 600 605 Arg Ala Met Ser Val Val Ser Ile Met Thr SerVal Ile Glu Glu Leu 610 615 620 Glu Glu Ser Lys Leu Lys Cys Pro Pro CysLeu Ile Ser Phe Ala Gln 625 630 635 640 Lys Tyr Leu Ile Trp Glu Cys CysPro Lys Trp Arg Lys Phe Lys Met 645 650 655 Ala Leu Phe Glu Leu Val ThrAsp Pro Phe Ala Glu Leu Thr Ile Thr 660 665 670 Leu Cys Ile Val Val AsnThr Val Phe Met Ala Met Glu His Tyr Pro 675 680 685 Met Thr Asp Ala PheAsp Ala Met Leu Gln Ala Gly Asn Ile Val Phe 690 695 700 Thr Val Phe PheThr Met Glu Met Ala Phe Lys Ile Ile Ala Phe Asp 705 710 715 720 Pro TyrTyr Tyr Phe Gln Lys Lys Trp Asn Ile Phe Asp Cys Val Ile 725 730 735 ValThr Val Ser Leu Leu Glu Leu Ser Ala Ser Lys Lys Gly Ser Leu 740 745 750Ser Val Leu Arg Thr Leu Arg Leu Leu Arg Val Phe Lys Leu Ala Lys 755 760765 Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys Ile Ile Gly Asn Ser Val 770775 780 Gly Ala Leu Gly Asn Leu Thr Phe Ile Leu Ala Ile Ile Val Phe Ile785 790 795 800 Phe Ala Leu Val Gly Lys Gln Leu Leu Ser Glu Asp Tyr GlyCys Arg 805 810 815 Lys Asp Gly Val Ser Val Trp Asn Gly Glu Lys Leu ArgTrp His Met 820 825 830 Cys Asp Phe Phe His Ser Phe Leu Val Val Phe ArgIle Leu Cys Gly 835 840 845 Glu Trp Ile Glu Asn Met Trp Val Cys Met GluVal Ser Gln Lys Ser 850 855 860 Ile Cys Leu Ile Leu Phe Leu Thr Val MetVal Leu Gly Asn Leu Val 865 870 875 880 Val Leu Asn Leu Phe Ile Ala LeuLeu Leu Asn Ser Phe Ser Ala Asp 885 890 895 Asn Leu Thr Ala Pro Glu AspAsp Gly Glu Val Asn Asn Leu Gln Leu 900 905 910 Ala Leu Ala Arg Ile GlnVal Leu Gly His Arg Ala Ser Arg Ala Ser 915 920 925 Ala Ser Tyr Ile SerSer His Cys Arg Phe His Trp Pro Lys Val Glu 930 935 940 Thr Gln Leu GlyMet Lys Pro Pro Leu Thr Ser Ser Glu Ala Lys Asn 945 950 955 960 His IleAla Thr Asp Ala Val Ser Ala Ala Val Gly Asn Leu Thr Lys 965 970 975 ProAla Leu Ser Ser Pro Lys Glu Asn His Gly Asp Phe Ile Thr Asp 980 985 990Pro Asn Val Trp Val Ser Val Pro Ile Ala Glu Gly Glu Ser Asp Leu 995 10001005 Asp Glu Leu Glu Glu Asp Met Glu Gln Ala Ser Gln Ser Ser Trp Gln1010 1015 1020 Glu Glu Asp Pro Lys Gly Gln Gln Glu Gln Leu Pro Gln ValGln Lys 1025 1030 1035 1040 Cys Glu Asn His Gln Ala Ala Arg Ser Pro AlaSer Met Met Ser Ser 1045 1050 1055 Glu Asp Leu Ala Pro Tyr Leu Gly GluSer Trp Lys Arg Lys Asp Ser 1060 1065 1070 Pro Gln Val Pro Ala Glu GlyVal Asp Asp Thr Ser Ser Ser Glu Gly 1075 1080 1085 Ser Thr Val Asp CysPro Asp Pro Glu Glu Ile Leu Arg Lys Ile Pro 1090 1095 1100 Glu Leu AlaHis Asp Leu Asp Glu Pro Asp Asp Cys Phe Arg Glu Gly 1105 1110 1115 1120Cys Thr Arg Arg Cys Pro Cys Cys Asn Val Asn Thr Ser Lys Ser Pro 11251130 1135 Trp Ala Thr Gly Trp Gln Val Arg Lys Thr Cys Tyr Arg Ile ValGlu 1140 1145 1150 His Ser Trp Phe Glu Ser Phe Ile Ile Phe Met Ile LeuLeu Ser Ser 1155 1160 1165 Gly Ala Leu Ala Phe Glu Asp Asn Tyr Leu GluGlu Lys Pro Arg Val 1170 1175 1180 Lys Ser Val Leu Glu Tyr Thr Asp ArgVal Phe Thr Phe Ile Phe Val 1185 1190 1195 1200 Phe Glu Met Leu Leu LysTrp Val Ala Tyr Gly Phe Lys Lys Tyr Phe 1205 1210 1215 Thr Asn Ala TrpCys Trp Leu Asp Phe Leu Ile Val Asn Ile Ser Leu 1220 1225 1230 Thr SerLeu Ile Ala Lys Ile Leu Glu Tyr Ser Asp Val Ala Ser Ile 1235 1240 1245Lys Ala Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser 12501255 1260 Arg Phe Glu Gly Met Arg Val Val Val Asp Ala Leu Val Gly AlaIle 1265 1270 1275 1280 Pro Ser Ile Met Asn Val Leu Leu Val Cys Leu IlePhe Trp Leu Ile 1285 1290 1295 Phe Ser Ile Met Gly Val Asn Leu Phe AlaGly Lys Phe Ser Lys Cys 1300 1305 1310 Val Asp Thr Arg Asn Asn Pro PheSer Asn Val Asn Ser Thr Met Val 1315 1320 1325 Asn Asn Lys Ser Glu CysHis Asn Gln Asn Ser Thr Gly His Phe Phe 1330 1335 1340 Trp Val Asn ValLys Val Asn Phe Asp Asn Val Ala Met Gly Tyr Leu 1345 1350 1355 1360 AlaLeu Leu Gln Val Ala Thr Phe Lys Gly Trp Met Asp Ile Met Tyr 1365 13701375 Ala Ala Val Asp Ser Gly Glu Ile Asn Ser Gln Pro Asn Trp Glu Asn1380 1385 1390 Asn Leu Tyr Met Tyr Leu Tyr Phe Val Val Phe Ile Ile PheGly Gly 1395 1400 1405 Phe Phe Thr Leu Asn Leu Phe Val Gly Val Ile IleAsp Asn Phe Asn 1410 1415 1420 Gln Gln Lys Lys Lys Leu Gly Gly Gln AspIle Phe Met Thr Glu Glu 1425 1430 1435 1440 Gln Lys Lys Tyr Tyr Asn AlaMet Lys Lys Leu Gly Ser Lys Lys Pro 1445 1450 1455 Gln Lys Pro Ile ProArg Pro Leu Asn Lys Tyr Gln Gly Phe Val Phe 1460 1465 1470 Asp Ile ValThr Arg Gln Ala Phe Asp Ile Ile Ile Met Val Leu Ile 1475 1480 1485 CysLeu Asn Met Ile Thr Met Met Val Glu Thr Asp Glu Gln Gly Glu 1490 14951500 Glu Lys Thr Lys Val Leu Gly Arg Ile Asn Gln Phe Phe Val Ala Val1505 1510 1515 1520 Phe Thr Gly Glu Cys Val Met Lys Met Phe Ala Leu ArgGln Tyr Tyr 1525 1530 1535 Phe Thr Asn Gly Trp Asn Val Phe Asp Phe IleVal Val Ile Leu Ser 1540 1545 1550 Ile Gly Ser Leu Leu Phe Ser Ala IleLeu Lys Ser Leu Glu Asn Tyr 1555 1560 1565 Phe Ser Pro Thr Leu Phe ArgVal Ile Arg Leu Ala Arg Ile Gly Arg 1570 1575 1580 Ile Leu Arg Leu IleArg Ala Ala Lys Gly Ile Arg Thr Leu Leu Phe 1585 1590 1595 1600 Ala LeuMet Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu 1605 1610 1615Phe Leu Val Met Phe Ile Tyr Ser Ile Phe Gly Met Ala Ser Phe Ala 16201625 1630 Asn Val Val Asp Glu Ala Gly Ile Asp Asp Met Phe Asn Phe LysThr 1635 1640 1645 Phe Gly Asn Ser Met Leu Cys Leu Phe Gln Ile Thr ThrSer Ala Gly 1650 1655 1660 Trp Asp Gly Leu Leu Ser Pro Ile Leu Asn ThrGly Pro Pro Tyr Cys 1665 1670 1675 1680 Asp Pro Asn Leu Pro Asn Ser AsnGly Ser Arg Gly Asn Cys Gly Ser 1685 1690 1695 Pro Ala Val Gly Ile IlePhe Phe Thr Thr Tyr Ile Ile Ile Ser Phe 1700 1705 1710 Leu Ile Val ValAsn Met Tyr Ile Ala Val Ile Leu Glu Asn Phe Asn 1715 1720 1725 Val AlaThr Glu Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp Phe Asp 1730 1735 1740Met Phe Tyr Glu Thr Trp Glu Lys Phe Asp Pro Glu Ala Thr Gln Phe 17451750 1755 1760 Ile Ala Phe Ser Ala Leu Ser Asp Phe Ala Asp Thr Leu SerGly Pro 1765 1770 1775 Leu Arg Ile Pro Lys Pro Asn Gln Asn Ile Leu IleGln Met Asp Leu 1780 1785 1790 Pro Leu Val Pro Gly Asp Lys Ile His CysLeu Asp Ile Leu Phe Ala 1795 1800 1805 Phe Thr Lys Asn Val Leu Gly GluSer Gly Glu Leu Asp Ser Leu Lys 1810 1815 1820 Thr Asn Met Glu Glu LysPhe Met Ala Thr Asn Leu Ser Lys Ala Ser 1825 1830 1835 1840 Tyr Glu ProIle Ala Thr Thr Leu Arg Trp Lys Gln Glu Asp Leu Ser 1845 1850 1855 AlaThr Val Ile Gln Lys Ala Tyr Arg Ser Tyr Met Leu His Arg Ser 1860 18651870 Leu Thr Leu Ser Asn Thr Leu His Val Pro Arg Ala Glu Glu Asp Gly1875 1880 1885 Val Ser Leu Pro Gly Glu Gly Tyr Ile Thr Phe Met Ala AsnSer Gly 1890 1895 1900 Leu Pro Asp Lys Ser Glu Thr Ala Ser Ala Thr SerPhe Pro Pro Ser 1905 1910 1915 1920 Tyr Asp Ser Val Thr Arg Gly Leu SerAsp Arg Ala Asn Ile Asn Pro 1925 1930 1935 Ser Ser Ser Met Gln Asn GluAsp Glu Val Ala Ala Lys Glu Gly Asn 1940 1945 1950 Ser Pro Gly Pro Gln1955 2573 base pairs nucleic acid single linear cDNA CDS 561..2126 3CTGGGAGAGA AAGCGTCTCG CCTAGCGACT CCCAGAGCTT TAAGCCGGGA AGGGACAAGC 60GTCAGGACAT CTCAGAATCC CGAACCTTCT AGGGAGGGAG GTTCTTACCT CCATGCTTCC 120CGTAGGAACC TAATCCCAAT TATTTAGCTG TATTTATAAT ACAAAATATG AATGTTAAAT 180GTACAAAATG CTTTCCCAGC ATGCCTGCAT CTCCTCCTAG AGTCCTGTTC CCAAGCCCTC 240TCTACTCTCA GTACTGTAGA AAAGAAATAA GCTTTACGTG AGAAACCCAG GCACTGGATC 300TTATCCAGGT GCTCACCTCA GAGTCTTTAG TGGGTGTAGC GCTGTGGTAG AGCATTTGGT 360TATAGATACA AACCCAGGGC AGGGAGACTG CAGTGGCCAT TCTCTCCCAG GCCAGACGTG 420CCCTGATCCT TCCCACAGAG ATGAGAAGGC TGGAACCAGA ACACTCAGGT TTTGGCTTCT 480CTTGGGGGAG GAGAGGTAAT CTTGTTACTT TAATAACATC AGTGTGTCCC TCTCCTCTAC 540TAGGAGGCCA GGACATCTTC ATG ACA GAA GAG CAG AAG AAG TAC TAC AAT 590 MetThr Glu Glu Gln Lys Lys Tyr Tyr Asn 1 5 10 GCC ATG AAG AAG CTG GGC TCCAAG AAA CCC CAG AAG CCC ATC CCA CGG 638 Ala Met Lys Lys Leu Gly Ser LysLys Pro Gln Lys Pro Ile Pro Arg 15 20 25 CCC CTG AAT AAG TAC CAA GGC TTCGTG TTT GAC ATC GTG ACC AGG CAA 686 Pro Leu Asn Lys Tyr Gln Gly Phe ValPhe Asp Ile Val Thr Arg Gln 30 35 40 GCC TTT GAC ATC ATC ATC ATG GTT CTCATC TGC CTC AAC ATG ATC ACC 734 Ala Phe Asp Ile Ile Ile Met Val Leu IleCys Leu Asn Met Ile Thr 45 50 55 ATG ATG GTG GAG ACC GAC GAG CAG GGC GAGGAG AAG ACG AAG GTT CTG 782 Met Met Val Glu Thr Asp Glu Gln Gly Glu GluLys Thr Lys Val Leu 60 65 70 GGC AGA ATC AAC CAG TTC TTT GTG GCC GTC TTCACG GGC GAG TGT GTG 830 Gly Arg Ile Asn Gln Phe Phe Val Ala Val Phe ThrGly Glu Cys Val 75 80 85 90 ATG AAG ATG TTC GCC CTG CGA CAG TAC TAC TTCACC AAC GGC TGG AAC 878 Met Lys Met Phe Ala Leu Arg Gln Tyr Tyr Phe ThrAsn Gly Trp Asn 95 100 105 GTG TTC GAC TTC ATA GTG GTG ATC CTG TCC ATTGGG AGT CTG CTG TTT 926 Val Phe Asp Phe Ile Val Val Ile Leu Ser Ile GlySer Leu Leu Phe 110 115 120 TCT GCA ATC CTT AAG TCA CTG GAA AAC TAC TTCTCC CCG ACG CTC TTC 974 Ser Ala Ile Leu Lys Ser Leu Glu Asn Tyr Phe SerPro Thr Leu Phe 125 130 135 CGG GTC ATC CGT CTG GCC AGG ATC GGC CGC ATCCTC AGG CTG ATC CGA 1022 Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile LeuArg Leu Ile Arg 140 145 150 GCA GCC AAG GGG ATT CGC ACG CTG CTC TTC GCCCTC ATG ATG TCC CTG 1070 Ala Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala LeuMet Met Ser Leu 155 160 165 170 CCC GCC CTC TTC AAC ATC GGC CTC CTC CTCTTC CTC GTC ATG TTC ATC 1118 Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu PheLeu Val Met Phe Ile 175 180 185 TAC TCC ATC TTC GGC ATG GCC AGC TTC GCTAAC GTC GTG GAC GAG GCC 1166 Tyr Ser Ile Phe Gly Met Ala Ser Phe Ala AsnVal Val Asp Glu Ala 190 195 200 GGC ATC GAC GAC ATG TTC AAC TTC AAG ACCTTT GGC AAC AGC ATG CTG 1214 Gly Ile Asp Asp Met Phe Asn Phe Lys Thr PheGly Asn Ser Met Leu 205 210 215 TGC CTG TTC CAG ATC ACC ACC TCG GCC GGCTGG GAC GGC CTC CTC AGC 1262 Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly TrpAsp Gly Leu Leu Ser 220 225 230 CCC ATC CTC AAC ACG GGG CCT CCC TAC TGCGAC CCC AAC CTG CCC AAC 1310 Pro Ile Leu Asn Thr Gly Pro Pro Tyr Cys AspPro Asn Leu Pro Asn 235 240 245 250 AGC AAC GGC TCC CGG GGG AAC TGC GGGAGC CCG GCG GTG GGC ATC ATC 1358 Ser Asn Gly Ser Arg Gly Asn Cys Gly SerPro Ala Val Gly Ile Ile 255 260 265 TTC TTC ACC ACC TAC ATC ATC ATC TCCTTC CTC ATC GTG GTC AAC ATG 1406 Phe Phe Thr Thr Tyr Ile Ile Ile Ser PheLeu Ile Val Val Asn Met 270 275 280 TAC ATC GCA GTG ATT CTG GAG AAC TTCAAC GTA GCC ACC GAG GAG AGC 1454 Tyr Ile Ala Val Ile Leu Glu Asn Phe AsnVal Ala Thr Glu Glu Ser 285 290 295 ACG GAG CCC CTG AGC GAG GAC GAC TTCGAC ATG TTC TAT GAG ACC TGG 1502 Thr Glu Pro Leu Ser Glu Asp Asp Phe AspMet Phe Tyr Glu Thr Trp 300 305 310 GAG AAG TTC GAC CCG GAG GCC ACC CAGTTC ATT GCC TTT TCT GCC CTC 1550 Glu Lys Phe Asp Pro Glu Ala Thr Gln PheIle Ala Phe Ser Ala Leu 315 320 325 330 TCA GAC TTC GCG GAC ACG CTC TCCGGC CCT CTT AGA ATC CCC AAA CCC 1598 Ser Asp Phe Ala Asp Thr Leu Ser GlyPro Leu Arg Ile Pro Lys Pro 335 340 345 AAC CAG AAT ATA TTA ATC CAG ATGGAC CTG CCG TTG GTC CCC GGG GAT 1646 Asn Gln Asn Ile Leu Ile Gln Met AspLeu Pro Leu Val Pro Gly Asp 350 355 360 AAG ATC CAC TGT CTG GAC ATC CTTTTT GCC TTC ACA AAG AAC GTC TTG 1694 Lys Ile His Cys Leu Asp Ile Leu PheAla Phe Thr Lys Asn Val Leu 365 370 375 GGA GAA TCC GGG GAG TTG GAC TCCCTG AAG ACC AAT ATG GAA GAG AAG 1742 Gly Glu Ser Gly Glu Leu Asp Ser LeuLys Thr Asn Met Glu Glu Lys 380 385 390 TTT ATG GCG ACC AAT CTC TCC AAAGCA TCC TAT GAA CCA ATA GCC ACC 1790 Phe Met Ala Thr Asn Leu Ser Lys AlaSer Tyr Glu Pro Ile Ala Thr 395 400 405 410 ACC CTC CGG TGG AAG CAG GAAGAC CTC TCA GCC ACA GTC ATT CAA AAG 1838 Thr Leu Arg Trp Lys Gln Glu AspLeu Ser Ala Thr Val Ile Gln Lys 415 420 425 GCC TAC CGG AGC TAC ATG CTGCAC CGC TCC TTG ACA CTC TCC AAC ACC 1886 Ala Tyr Arg Ser Tyr Met Leu HisArg Ser Leu Thr Leu Ser Asn Thr 430 435 440 CTG CAT GTG CCC AGG GCT GAGGAG GAT GGC GTG TCA CTT CCC GGG GAA 1934 Leu His Val Pro Arg Ala Glu GluAsp Gly Val Ser Leu Pro Gly Glu 445 450 455 GGC TAC ATT ACA TTC ATG GCAAAC AGT GGA CTC CCG GAC AAA TCA GAA 1982 Gly Tyr Ile Thr Phe Met Ala AsnSer Gly Leu Pro Asp Lys Ser Glu 460 465 470 ACT GCC TCT GCT ACG TCT TTCCCG CCA TCC TAT GAC AGT GTC ACC AGG 2030 Thr Ala Ser Ala Thr Ser Phe ProPro Ser Tyr Asp Ser Val Thr Arg 475 480 485 490 GGC CTG AGT GAC CGG GCCAAC ATT AAC CCA TCT AGC TCA ATG CAA AAT 2078 Gly Leu Ser Asp Arg Ala AsnIle Asn Pro Ser Ser Ser Met Gln Asn 495 500 505 GAA GAT GAG GTC GCT GCTAAG GAA GGA AAC AGC CCT GGA CCT CAG TGAAGGCA2133 Glu Asp Glu Val Ala AlaLys Glu Gly Asn Ser Pro Gly Pro Gln 510 515 520 CAGGCATGCA CAGGGCAGGTTCCAATGTCT TTCTCTGCTG TACTAACTCC TTCCCTCTGG 2193 AGGTGGCACC AACCTCCAGCCTCCACCAAT GCATGTCACT GGTCATGGTG TCAGAACTGA 2253 ATGGGGACAT CCTTGAGAAAGCCCCCACCC CAATAGGAAT CAAAAGCCAA GGATACTCCT 2313 CCATTCTGAC GTCCCTTCCGAGTTCCCAGA AGATGTCATT GCTCCCTTCT GTTTGTGACC 2373 AGAGACGTGA TTCACCAACTTCTCGGAGCC AGAGACACAT AGCAAAGACT TTTCTGCTGG 2433 TGTCGGGCAG TCTTAGAGAAGTCACGTAGG GGTTGGTACT GAGAATTAGG GTTTGCATGA 2493 CTGCATGCTC ACAGCTGCCGGACAATACCT GTGAGTCGGC CATTAAAATT AATATTTTTA 2553 AAGTTAAAAA AAAAAAAAAA2573 521 amino acids amino acid linear protein 4 Met Thr Glu Glu Gln LysLys Tyr Tyr Asn Ala Met Lys Lys Leu Gly 1 5 10 15 Ser Lys Lys Pro GlnLys Pro Ile Pro Arg Pro Leu Asn Lys Tyr Gln 20 25 30 Gly Phe Val Phe AspIle Val Thr Arg Gln Ala Phe Asp Ile Ile Ile 35 40 45 Met Val Leu Ile CysLeu Asn Met Ile Thr Met Met Val Glu Thr Asp 50 55 60 Glu Gln Gly Glu GluLys Thr Lys Val Leu Gly Arg Ile Asn Gln Phe 65 70 75 80 Phe Val Ala ValPhe Thr Gly Glu Cys Val Met Lys Met Phe Ala Leu 85 90 95 Arg Gln Tyr TyrPhe Thr Asn Gly Trp Asn Val Phe Asp Phe Ile Val 100 105 110 Val Ile LeuSer Ile Gly Ser Leu Leu Phe Ser Ala Ile Leu Lys Ser 115 120 125 Leu GluAsn Tyr Phe Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala 130 135 140 ArgIle Gly Arg Ile Leu Arg Leu Ile Arg Ala Ala Lys Gly Ile Arg 145 150 155160 Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile 165170 175 Gly Leu Leu Leu Phe Leu Val Met Phe Ile Tyr Ser Ile Phe Gly Met180 185 190 Ala Ser Phe Ala Asn Val Val Asp Glu Ala Gly Ile Asp Asp MetPhe 195 200 205 Asn Phe Lys Thr Phe Gly Asn Ser Met Leu Cys Leu Phe GlnIle Thr 210 215 220 Thr Ser Ala Gly Trp Asp Gly Leu Leu Ser Pro Ile LeuAsn Thr Gly 225 230 235 240 Pro Pro Tyr Cys Asp Pro Asn Leu Pro Asn SerAsn Gly Ser Arg Gly 245 250 255 Asn Cys Gly Ser Pro Ala Val Gly Ile IlePhe Phe Thr Thr Tyr Ile 260 265 270 Ile Ile Ser Phe Leu Ile Val Val AsnMet Tyr Ile Ala Val Ile Leu 275 280 285 Glu Asn Phe Asn Val Ala Thr GluGlu Ser Thr Glu Pro Leu Ser Glu 290 295 300 Asp Asp Phe Asp Met Phe TyrGlu Thr Trp Glu Lys Phe Asp Pro Glu 305 310 315 320 Ala Thr Gln Phe IleAla Phe Ser Ala Leu Ser Asp Phe Ala Asp Thr 325 330 335 Leu Ser Gly ProLeu Arg Ile Pro Lys Pro Asn Gln Asn Ile Leu Ile 340 345 350 Gln Met AspLeu Pro Leu Val Pro Gly Asp Lys Ile His Cys Leu Asp 355 360 365 Ile LeuPhe Ala Phe Thr Lys Asn Val Leu Gly Glu Ser Gly Glu Leu 370 375 380 AspSer Leu Lys Thr Asn Met Glu Glu Lys Phe Met Ala Thr Asn Leu 385 390 395400 Ser Lys Ala Ser Tyr Glu Pro Ile Ala Thr Thr Leu Arg Trp Lys Gln 405410 415 Glu Asp Leu Ser Ala Thr Val Ile Gln Lys Ala Tyr Arg Ser Tyr Met420 425 430 Leu His Arg Ser Leu Thr Leu Ser Asn Thr Leu His Val Pro ArgAla 435 440 445 Glu Glu Asp Gly Val Ser Leu Pro Gly Glu Gly Tyr Ile ThrPhe Met 450 455 460 Ala Asn Ser Gly Leu Pro Asp Lys Ser Glu Thr Ala SerAla Thr Ser 465 470 475 480 Phe Pro Pro Ser Tyr Asp Ser Val Thr Arg GlyLeu Ser Asp Arg Ala 485 490 495 Asn Ile Asn Pro Ser Ser Ser Met Gln AsnGlu Asp Glu Val Ala Ala 500 505 510 Lys Glu Gly Asn Ser Pro Gly Pro Gln515 520 7052 base pairs nucleic acid single linear cDNA CDS 204..6602 5TAGCTTGCTT CTGCTAATGC TACCCCAGGC CTTTAGACAG AGAACAGATG GCAGATGGAG 60TTTCTTATTG CCATGCGCAA ACGCTGAGCC CACCTCATGA TCCCGGACCC CATGGTTTTC 120AGTAGACAAC CTGGGCTAAG AAGAGATCTC CGACCTTATA GAGCAGCAAA GAGTGTAAAT 180TCTTCCCCAA GAAGAATGAG AAG ATG GAG CTC CCC TTT GCG TCC GTG GGA 230 MetGlu Leu Pro Phe Ala Ser Val Gly 1 5 ACT ACC AAT TTC AGA CGG TTC ACT CCAGAG TCA CTG GCA GAG ATC GAG 278 Thr Thr Asn Phe Arg Arg Phe Thr Pro GluSer Leu Ala Glu Ile Glu 10 15 20 25 AAG CAG ATT GCT GCT CAC CGG GCA GCCAAG AAG GCC AGA ACC AAG CAC 326 Lys Gln Ile Ala Ala His Arg Ala Ala LysLys Ala Arg Thr Lys His 30 35 40 AGA GGA CAG GAG GAC AAG GGC GAG AAG CCCAGG CCT CAG CTG GAC TTG 374 Arg Gly Gln Glu Asp Lys Gly Glu Lys Pro ArgPro Gln Leu Asp Leu 45 50 55 AAA GAC TGT AAC CAG CTG CCC AAG TTC TAT GGTGAG CTC CCA GCA GAA 422 Lys Asp Cys Asn Gln Leu Pro Lys Phe Tyr Gly GluLeu Pro Ala Glu 60 65 70 CTG GTC GGG GAG CCC CTG GAG GAC CTA GAC CCT TTCTAC AGC ACA CAC 470 Leu Val Gly Glu Pro Leu Glu Asp Leu Asp Pro Phe TyrSer Thr His 75 80 85 CGG ACA TTC ATG GTG TTG AAT AAA AGC AGG ACC ATT TCCAGA TTC AGT 518 Arg Thr Phe Met Val Leu Asn Lys Ser Arg Thr Ile Ser ArgPhe Ser 90 95 100 105 GCC ACT TGG GCC CTG TGG CTC TTC AGT CCC TTC AACCTG ATC AGA AGA 566 Ala Thr Trp Ala Leu Trp Leu Phe Ser Pro Phe Asn LeuIle Arg Arg 110 115 120 ACA GCC ATC AAA GTG TCT GTC CAT TCC TGG TTC TCCATA TTC ATC ACC 614 Thr Ala Ile Lys Val Ser Val His Ser Trp Phe Ser IlePhe Ile Thr 125 130 135 ATC ACT ATT TTG GTC AAC TGC GTG TGC ATG ACC CGAACT GAT CTT CCA 662 Ile Thr Ile Leu Val Asn Cys Val Cys Met Thr Arg ThrAsp Leu Pro 140 145 150 GAG AAA GTC GAG TAC GTC TTC ACT GTC ATT TAC ACCTTC GAG GCT CTG 710 Glu Lys Val Glu Tyr Val Phe Thr Val Ile Tyr Thr PheGlu Ala Leu 155 160 165 ATT AAG ATA CTG GCA AGA GGG TTT TGT CTA AAT GAGTTC ACT TAT CTT 758 Ile Lys Ile Leu Ala Arg Gly Phe Cys Leu Asn Glu PheThr Tyr Leu 170 175 180 185 CGA GAT CCG TGG AAC TGG CTG GAC TTC AGT GTCATT ACC TTG GCG TAT 806 Arg Asp Pro Trp Asn Trp Leu Asp Phe Ser Val IleThr Leu Ala Tyr 190 195 200 GTG GGT GCA GCG ATA GAC CTC CGA GGA ATC TCAGGC CTG CGG ACA TTC 854 Val Gly Ala Ala Ile Asp Leu Arg Gly Ile Ser GlyLeu Arg Thr Phe 205 210 215 CGA GTT CTC AGA GCC CTG AAA ACT GTT TCT GTGATC CCA GGA CTG AAG 902 Arg Val Leu Arg Ala Leu Lys Thr Val Ser Val IlePro Gly Leu Lys 220 225 230 GTC ATC GTG GGA GCC CTG ATC CAC TCA GTG AGGAAG CTG GCC GAC GTG 950 Val Ile Val Gly Ala Leu Ile His Ser Val Arg LysLeu Ala Asp Val 235 240 245 ACT ATC CTC ACA GTC TTC TGC CTG AGC GTC TTCGCC TTG GTG GGC CTG 998 Thr Ile Leu Thr Val Phe Cys Leu Ser Val Phe AlaLeu Val Gly Leu 250 255 260 265 CAG CTC TTT AAG GGG AAC CTT AAG AAC AAATGC ATC AGG AAC GGA ACA 1046 Gln Leu Phe Lys Gly Asn Leu Lys Asn Lys CysIle Arg Asn Gly Thr 270 275 280 GAT CCC CAC AAG GCT GAC AAC CTC TCA TCTGAA ATG GCA GAA TAC ATC 1094 Asp Pro His Lys Ala Asp Asn Leu Ser Ser GluMet Ala Glu Tyr Ile 285 290 295 TTC ATC AAG CCT GGT ACT ACG GAT CCC TTACTG TGC GGC AAT GGG TCT 1142 Phe Ile Lys Pro Gly Thr Thr Asp Pro Leu LeuCys Gly Asn Gly Ser 300 305 310 GAT GCT GGT CAC TGC CCT GGA GGC TAT GTCTGC CTG AAA ACT CCT GAC 1190 Asp Ala Gly His Cys Pro Gly Gly Tyr Val CysLeu Lys Thr Pro Asp 315 320 325 AAC CCG GAT TTT AAC TAC ACC AGC TTT GATTCC TTT GCG TGG GCA TTC 1238 Asn Pro Asp Phe Asn Tyr Thr Ser Phe Asp SerPhe Ala Trp Ala Phe 330 335 340 345 CTC TCA CTG TTC CGC CTC ATG ACG CAGGAC TCC TGG GAG CGC CTG TAC 1286 Leu Ser Leu Phe Arg Leu Met Thr Gln AspSer Trp Glu Arg Leu Tyr 350 355 360 CAG CAG ACA CTC CGG GCT TCT GGG AAAATG TAC ATG GTC TTT TTC GTG 1334 Gln Gln Thr Leu Arg Ala Ser Gly Lys MetTyr Met Val Phe Phe Val 365 370 375 CTG GTT ATT TTC CTT GGA TCG TTC TACCTG GTC AAT TTG ATC TTG GCC 1382 Leu Val Ile Phe Leu Gly Ser Phe Tyr LeuVal Asn Leu Ile Leu Ala 380 385 390 GTG GTC ACC ATG GCG TAT GAA GAG CAGAGC CAG GCA ACA ATT GCA GAA 1430 Val Val Thr Met Ala Tyr Glu Glu Gln SerGln Ala Thr Ile Ala Glu 395 400 405 ATC GAA GCC AAG GAA AAA AAG TTC CAGGAA GCC CTT GAG GTG CTG CAG 1478 Ile Glu Ala Lys Glu Lys Lys Phe Gln GluAla Leu Glu Val Leu Gln 410 415 420 425 AAG GAA CAG GAG GTG CTG GCA GCCCTG GGG ATT GAC ACG ACC TCG CTC 1526 Lys Glu Gln Glu Val Leu Ala Ala LeuGly Ile Asp Thr Thr Ser Leu 430 435 440 CAG TCC CAC AGT GGA TCA CCC TTAGCC TCC AAA AAC GCC AAT GAG AGA 1574 Gln Ser His Ser Gly Ser Pro Leu AlaSer Lys Asn Ala Asn Glu Arg 445 450 455 AGA CCC AGG GTG AAA TCA AGG GTGTCA GAG GGC TCC ACG GAT GAC AAC 1622 Arg Pro Arg Val Lys Ser Arg Val SerGlu Gly Ser Thr Asp Asp Asn 460 465 470 AGG TCA CCC CAA TCT GAC CCT TACAAC CAG CGC AGG ATG TCT TTC CTA 1670 Arg Ser Pro Gln Ser Asp Pro Tyr AsnGln Arg Arg Met Ser Phe Leu 475 480 485 GGC CTG TCT TCA GGA AGA CGC AGGGCT AGC CAC GGC AGT GTG TTC CAC 1718 Gly Leu Ser Ser Gly Arg Arg Arg AlaSer His Gly Ser Val Phe His 490 495 500 505 TTC CGA GCG CCC AGC CAA GACATC TCA TTT CCT GAC GGG ATC ACC CCT 1766 Phe Arg Ala Pro Ser Gln Asp IleSer Phe Pro Asp Gly Ile Thr Pro 510 515 520 GAT GAT GGG GTC TTT CAC GGAGAC CAG GAA AGC CGT CGA GGT TCC ATA 1814 Asp Asp Gly Val Phe His Gly AspGln Glu Ser Arg Arg Gly Ser Ile 525 530 535 TTG CTG GGC AGG GGT GCT GGGCAG ACA GGT CCA CTC CCC AGG AGC CCA 1862 Leu Leu Gly Arg Gly Ala Gly GlnThr Gly Pro Leu Pro Arg Ser Pro 540 545 550 CTG CCT CAG TCC CCC AAC CCTGGC CGT AGA CAT GGA GAA GAG GGA CAG 1910 Leu Pro Gln Ser Pro Asn Pro GlyArg Arg His Gly Glu Glu Gly Gln 555 560 565 CTC GGA GTG CCC ACT GGT GAGCTT ACC GCT GGA GCG CCT GAA GGC CCG 1958 Leu Gly Val Pro Thr Gly Glu LeuThr Ala Gly Ala Pro Glu Gly Pro 570 575 580 585 GCA CTC GAC ACT ACA GGGCAG AAG AGC TTC CTG TCT GCG GGC TAC TTG 2006 Ala Leu Asp Thr Thr Gly GlnLys Ser Phe Leu Ser Ala Gly Tyr Leu 590 595 600 AAC GAA CCT TTC CGA GCACAG AGG GCC ATG AGC GTT GTC AGT ATC ATG 2054 Asn Glu Pro Phe Arg Ala GlnArg Ala Met Ser Val Val Ser Ile Met 605 610 615 ACT TCT GTC ATT GAG GAGCTT GAA GAG TCT AAG CTG AAG TGC CCA CCC 2102 Thr Ser Val Ile Glu Glu LeuGlu Glu Ser Lys Leu Lys Cys Pro Pro 620 625 630 TGC TTG ATC AGC TTC GCTCAG AAG TAT CTG ATC TGG GAG TGC TGC CCC 2150 Cys Leu Ile Ser Phe Ala GlnLys Tyr Leu Ile Trp Glu Cys Cys Pro 635 640 645 AAG TGG AGG AAG TTC AAGATG GCG CTG TTC GAG CTG GTG ACT GAC CCC 2198 Lys Trp Arg Lys Phe Lys MetAla Leu Phe Glu Leu Val Thr Asp Pro 650 655 660 665 TTC GCA GAG CTT ACCATC ACC CTC TGC ATC GTG GTG AAC ACC GTC TTC 2246 Phe Ala Glu Leu Thr IleThr Leu Cys Ile Val Val Asn Thr Val Phe 670 675 680 ATG GCC ATG GAG CACTAC CCC ATG ACC GAT GCC TTC GAT GCC ATG CTT 2294 Met Ala Met Glu His TyrPro Met Thr Asp Ala Phe Asp Ala Met Leu 685 690 695 CAA GCC GGC AAC ATTGTC TTC ACC GTG TTT TTC ACA ATG GAG ATG GCC 2342 Gln Ala Gly Asn Ile ValPhe Thr Val Phe Phe Thr Met Glu Met Ala 700 705 710 TTC AAG ATC ATT GCCTTC GAC CCC TAC TAT TAC TTC CAG AAG AAG TGG 2390 Phe Lys Ile Ile Ala PheAsp Pro Tyr Tyr Tyr Phe Gln Lys Lys Trp 715 720 725 AAT ATC TTC GAC TGTGTC ATC GTC ACC GTG AGC CTT CTG GAG CTG AGT 2438 Asn Ile Phe Asp Cys ValIle Val Thr Val Ser Leu Leu Glu Leu Ser 730 735 740 745 GCA TCC AAG AAGGGC AGC CTG TCT GTG CTC CGT TCC TTA CGC TTG GCA 2486 Ala Ser Lys Lys GlySer Leu Ser Val Leu Arg Ser Leu Arg Leu Ala 750 755 760 CTC GAC ACT ACAGGG CAG AAG AGC TTC CTG TCT GCG GGC TAC TTG AAC 2534 Leu Asp Thr Thr GlyGln Lys Ser Phe Leu Ser Ala Gly Tyr Leu Asn 765 770 775 GAA CCT TTC CGAGCA CAG AGG GCC ATG AGC GTT GTC AGT ATC ATG ACT 2582 Glu Pro Phe Arg AlaGln Arg Ala Met Ser Val Val Ser Ile Met Thr 780 785 790 TCT GTC ATT GAGGAG CTT GAA GAG TCT AAG CTG AAG TGC CCA CCC TGC 2630 Ser Val Ile Glu GluLeu Glu Glu Ser Lys Leu Lys Cys Pro Pro Cys 795 800 805 TTG ATC AGC TTCGCT CAG AAG TAT CTG ATC TGG GAG TGC TGC CCC AAG 2678 Leu Ile Ser Phe AlaGln Lys Tyr Leu Ile Trp Glu Cys Cys Pro Lys 810 815 820 825 TGG AGG AAGTTC AAG ATG GCG CTG TTC GAG CTG GTG ACT GAC CCC TTC 2726 Trp Arg Lys PheLys Met Ala Leu Phe Glu Leu Val Thr Asp Pro Phe 830 835 840 GCA GAG CTTACC ATC ACC CTC TGC ATC GTG GTG AAC ACC GTC TTC ATG 2774 Ala Glu Leu ThrIle Thr Leu Cys Ile Val Val Asn Thr Val Phe Met 845 850 855 GCC ATG GAGCAC TAC CCC ATG ACC GAT GCC TTC GAT GCC ATG CTT CAA 2822 Ala Met Glu HisTyr Pro Met Thr Asp Ala Phe Asp Ala Met Leu Gln 860 865 870 GCC GGC AACATT GTC TTC ACC GTG TTT TTC ACA ATG GAG ATG GCC TTC 2870 Ala Gly Asn IleVal Phe Thr Val Phe Phe Thr Met Glu Met Ala Phe 875 880 885 AAG ATC ATTGCC TTC GAC CCC TAC TAT TAC TTC CAG AAG AAG TGG AAT 2918 Lys Ile Ile AlaPhe Asp Pro Tyr Tyr Tyr Phe Gln Lys Lys Trp Asn 890 895 900 905 ATC TTCGAC TGT GTC ATC GTC ACC GTG AGC CTT CTG GAG CTG AGT GCA 2966 Ile Phe AspCys Val Ile Val Thr Val Ser Leu Leu Glu Leu Ser Ala 910 915 920 TCC AAGAAG GGC AGC CTG TCT GTG CTC CGT TCC TTA CGC TTG CTG CGG 3014 Ser Lys LysGly Ser Leu Ser Val Leu Arg Ser Leu Arg Leu Leu Arg 925 930 935 GTC TTCAAG CTG GCC AAG TCC TGG CCC ACC CTG AAC ACC CTC ATC AAG 3062 Val Phe LysLeu Ala Lys Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys 940 945 950 ATC ATCGGG AAC TCA GTG GGG GCC CTG GGC AAC CTG ACC TTT ATC CTG 3110 Ile Ile GlyAsn Ser Val Gly Ala Leu Gly Asn Leu Thr Phe Ile Leu 955 960 965 GCC ATCATC GTC TTC ATC TTC GCC CTG GTC GGA AAG CAG CTT CTC TCA 3158 Ala Ile IleVal Phe Ile Phe Ala Leu Val Gly Lys Gln Leu Leu Ser 970 975 980 985 GAGGAC TAC GGG TGC CGC AAG GAC GGC GTC TCC GTG TGG AAC GGC GAG 3206 Glu AspTyr Gly Cys Arg Lys Asp Gly Val Ser Val Trp Asn Gly Glu 990 995 1000 AAGCTC CGC TGG CAC ATG TGT GAC TTC TTC CAT TCC TTC CTG GTC GTC 3254 Lys LeuArg Trp His Met Cys Asp Phe Phe His Ser Phe Leu Val Val 1005 1010 1015TTC CGA ATC CTC TGC GGG GAG TGG ATC GAG AAC ATG TGG GTC TGC ATG 3302 PheArg Ile Leu Cys Gly Glu Trp Ile Glu Asn Met Trp Val Cys Met 1020 10251030 GAG GTC AGC CAG AAA TCC ATC TGC CTC ATC CTC TTC TTG ACT GTG ATG3350 Glu Val Ser Gln Lys Ser Ile Cys Leu Ile Leu Phe Leu Thr Val Met1035 1040 1045 GTG CTG GGC AAC CTA GTG GTG CTC AAC CTT TTC ATC GCT TTACTG CTG 3398 Val Leu Gly Asn Leu Val Val Leu Asn Leu Phe Ile Ala Leu LeuLeu 1050 1055 1060 1065 AAC TCC TTC AGC GCG GAC AAC CTC ACG GCT CCA GAGGAT GAC GGG GAG 3446 Asn Ser Phe Ser Ala Asp Asn Leu Thr Ala Pro Glu AspAsp Gly Glu 1070 1075 1080 GTG AAC AAC TTG CAG TTA GCA CTG GCC AGG ATCCAG GTA CTT GGC CAT 3494 Val Asn Asn Leu Gln Leu Ala Leu Ala Arg Ile GlnVal Leu Gly His 1085 1090 1095 CGG GCC AGC AGG GCC ATC GCC AGT TAC ATCAGC AGC CAC TGC CGA TTC 3542 Arg Ala Ser Arg Ala Ile Ala Ser Tyr Ile SerSer His Cys Arg Phe 1100 1105 1110 CGC TGG CCC AAG GTG GAG ACC CAG CTGGGC ATG AAG CCC CCA CTC ACC 3590 Arg Trp Pro Lys Val Glu Thr Gln Leu GlyMet Lys Pro Pro Leu Thr 1115 1120 1125 AGC TCA GAG GCC AAG AAC CAC ATTGCC ACT GAT GCT GTC AGT GCT GCA 3638 Ser Ser Glu Ala Lys Asn His Ile AlaThr Asp Ala Val Ser Ala Ala 1130 1135 1140 1145 GTG GGG AAC CTG ACA AAGCCA GCT CTC AGT AGC CCC AAG GAG AAT CAC 3686 Val Gly Asn Leu Thr Lys ProAla Leu Ser Ser Pro Lys Glu Asn His 1150 1155 1160 GGG GAC TTC ATC ACTGAT CCC AAC GTG TGG GTC TCT GTG CCC ATT GCT 3734 Gly Asp Phe Ile Thr AspPro Asn Val Trp Val Ser Val Pro Ile Ala 1165 1170 1175 GAG GGG GAA TCTGAC CTC GAC GAG CTC GAG GAA GAT ATG GAG CAG GCT 3782 Glu Gly Glu Ser AspLeu Asp Glu Leu Glu Glu Asp Met Glu Gln Ala 1180 1185 1190 TCG CAG AGCTCC TGG CAG GAA GAG GAC CCC AAG GGA CAG CAG GAG CAG 3830 Ser Gln Ser SerTrp Gln Glu Glu Asp Pro Lys Gly Gln Gln Glu Gln 1195 1200 1205 TTG CCACAA GTC CAA AAG TGT GAA AAC CAC CAG GCA GCC AGA AGC CCA 3878 Leu Pro GlnVal Gln Lys Cys Glu Asn His Gln Ala Ala Arg Ser Pro 1210 1215 1220 1225GCC TCC ATG ATG TCC TCT GAG GAC CTG GCT CCA TAC CTG GGT GAG AGC 3926 AlaSer Met Met Ser Ser Glu Asp Leu Ala Pro Tyr Leu Gly Glu Ser 1230 12351240 TGG AAG AGG AAG GAT AGC CCT CAG GTC CCT GCC GAG GGA GTG GAT GAC3974 Trp Lys Arg Lys Asp Ser Pro Gln Val Pro Ala Glu Gly Val Asp Asp1245 1250 1255 ACG AGC TCC TCT GAG GGC AGC ACG GTG GAC TGC CCG GAC CCAGAG GAA 4022 Thr Ser Ser Ser Glu Gly Ser Thr Val Asp Cys Pro Asp Pro GluGlu 1260 1265 1270 ATC CTG AGG AAG ATC CCC GAG CTG GCA GAT GAC CTG GACGAG CCC GAT 4070 Ile Leu Arg Lys Ile Pro Glu Leu Ala Asp Asp Leu Asp GluPro Asp 1275 1280 1285 GAC TGT TTC ACA GAA GGC TGC ACT CGC CGC TGT CCCTGC TGC AAC GTG 4118 Asp Cys Phe Thr Glu Gly Cys Thr Arg Arg Cys Pro CysCys Asn Val 1290 1295 1300 1305 AAT ACT AGC AAG TCT CCT TGG GCC ACA GGCTGG CAG GTG CGC AAG ACC 4166 Asn Thr Ser Lys Ser Pro Trp Ala Thr Gly TrpGln Val Arg Lys Thr 1310 1315 1320 TGC TAC CGC ATC GTG GAG CAC AGC TGGTTT GAG AGT TTC ATC ATC TTC 4214 Cys Tyr Arg Ile Val Glu His Ser Trp PheGlu Ser Phe Ile Ile Phe 1325 1330 1335 ATG ATC CTG CTC AGC AGT GGA GCGCTG GCC TTT GAG GAT AAC TAC CTG 4262 Met Ile Leu Leu Ser Ser Gly Ala LeuAla Phe Glu Asp Asn Tyr Leu 1340 1345 1350 GAA GAG AAA CCC CGA GTG AAGTCC GTG CTG GAG TAC ACT GAC CGA GTG 4310 Glu Glu Lys Pro Arg Val Lys SerVal Leu Glu Tyr Thr Asp Arg Val 1355 1360 1365 TTC ACC TTC ATC TTC GTCTTT GAG ATG CTG CTC AAG TGG GTA GCC TAT 4358 Phe Thr Phe Ile Phe Val PheGlu Met Leu Leu Lys Trp Val Ala Tyr 1370 1375 1380 1385 GGC TTC AAA AAGTAT TTC ACC AAT GCC TGG TGC TGG CTG GAC TTC CTC 4406 Gly Phe Lys Lys TyrPhe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu 1390 1395 1400 ATT GTG AACATC TCC CTG ACA AGC CTC ATA GCG AAG ATC CTT GAG TAT 4454 Ile Val Asn IleSer Leu Thr Ser Leu Ile Ala Lys Ile Leu Glu Tyr 1405 1410 1415 TCC GACGTG GCG TCC ATC AAA GCC CTT CGG ACT CTC CGT GCC CTC CGA 4502 Ser Asp ValAla Ser Ile Lys Ala Leu Arg Thr Leu Arg Ala Leu Arg 1420 1425 1430 CCGCTG CGG GCT CTG TCT CGA TTC GAA GGC ATG AGG GTA GTG GTG GAT 4550 Pro LeuArg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asp 1435 1440 1445GCC CTC GTG GGC GCC ATC CCC TCC ATC ATG AAC GTC CTC CTC GTC TGC 4598 AlaLeu Val Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys 1450 14551460 1465 CTC ATC TTC TGG CTC ATC TTC AGC ATC ATG GGC GTG AAC CTC TTCGCC 4646 Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala1470 1475 1480 GGG AAA TTT TCG AAG TGC GTC GAC ACC AGA AAT AAC CCA TTTTCC AAC 4694 Gly Lys Phe Ser Lys Cys Val Asp Thr Arg Asn Asn Pro Phe SerAsn 1485 1490 1495 GTG AAT TCG ACG ATG GTG AAT AAC AAG TCC GAG TGT CACAAT CAA AAC 4742 Val Asn Ser Thr Met Val Asn Asn Lys Ser Glu Cys His AsnGln Asn 1500 1505 1510 AGC ACC GGC CAC TTC TTC TGG GTC AAC GTC AAA GTCAAC TTC GAC AAC 4790 Ser Thr Gly His Phe Phe Trp Val Asn Val Lys Val AsnPhe Asp Asn 1515 1520 1525 GTC GCT ATG GGC TAC CTC GCA CTT CTT CAG GTGGCA ACC TTC AAA GGC 4838 Val Ala Met Gly Tyr Leu Ala Leu Leu Gln Val AlaThr Phe Lys Gly 1530 1535 1540 1545 TGG ATG GAC ATA ATG TAT GCA GCT GTTGAT TCC GGA GAG ATC AAC AGT 4886 Trp Met Asp Ile Met Tyr Ala Ala Val AspSer Gly Glu Ile Asn Ser 1550 1555 1560 CAG CCT AAC TGG GAG AAC AAC TTGTAC ATG TAC CTG TAC TTC GTC GTT 4934 Gln Pro Asn Trp Glu Asn Asn Leu TyrMet Tyr Leu Tyr Phe Val Val 1565 1570 1575 TTC ATC ATT TTC GGT GGC TTCTTC ACG CTG AAT CTC TTT GTT GGG GTC 4982 Phe Ile Ile Phe Gly Gly Phe PheThr Leu Asn Leu Phe Val Gly Val 1580 1585 1590 ATA ATC GAC AAC TTC AACCAA CAG AAA AAA AAG CTA GGA GGC CAG GAC 5030 Ile Ile Asp Asn Phe Asn GlnGln Lys Lys Lys Leu Gly Gly Gln Asp 1595 1600 1605 ATC TTC ATG ACA GAAGAG CAG AAG AAG TAC TAC AAT GCC ATG AAG AAG 5078 Ile Phe Met Thr Glu GluGln Lys Lys Tyr Tyr Asn Ala Met Lys Lys 1610 1615 1620 1625 CTG GGC TCCAAG AAA CCC CAG AAG CCC ATC CCA CGG CCC CTG AAT AAG 5126 Leu Gly Ser LysLys Pro Gln Lys Pro Ile Pro Arg Pro Leu Asn Lys 1630 1635 1640 TAC CAAGGC TTC GTG TTT GAC ATC GTG ACC AGG CAA GCC TTT GAC ATC 5174 Tyr Gln GlyPhe Val Phe Asp Ile Val Thr Arg Gln Ala Phe Asp Ile 1645 1650 1655 ATCATC ATG GTT CTC ATC TGC CTC AAC ATG ATC ACC ATG ATG GTG GAG 5222 Ile IleMet Val Leu Ile Cys Leu Asn Met Ile Thr Met Met Val Glu 1660 1665 1670ACC GAC GAG CAG GGC GAG GAG AAG ACG AAG GTT CTG GGC AGA ATC AAC 5270 ThrAsp Glu Gln Gly Glu Glu Lys Thr Lys Val Leu Gly Arg Ile Asn 1675 16801685 CAG TTC TTT GTG GCC GTC TTC ACG GGC GAG TGT GTG ATG AAG ATG TTC5318 Gln Phe Phe Val Ala Val Phe Thr Gly Glu Cys Val Met Lys Met Phe1690 1695 1700 1705 GCC CTG CGA CAG TAC TAC TTC ACC AAC GGC TGG AAC GTGTTC GAC TTC 5366 Ala Leu Arg Gln Tyr Tyr Phe Thr Asn Gly Trp Asn Val PheAsp Phe 1710 1715 1720 ATA GTG GTG ATC CTG TCC ATT GGG AGT CTG CTG TTTTCT GCA ATC CTT 5414 Ile Val Val Ile Leu Ser Ile Gly Ser Leu Leu Phe SerAla Ile Leu 1725 1730 1735 AAG TCA CTG GAA AAC TAC TTC TCC CCG ACG CTCTTC CGG GTC ATC CGT 5462 Lys Ser Leu Glu Asn Tyr Phe Ser Pro Thr Leu PheArg Val Ile Arg 1740 1745 1750 CTG GCC AGG ATC GGC CGC ATC CTC AGG CTGATC CGA GCA GCC AAG GGG 5510 Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu IleArg Ala Ala Lys Gly 1755 1760 1765 ATT CGC ACG CTG CTC TTC GCC CTC ATGATG TCC CTG CCC GCC CTC TTC 5558 Ile Arg Thr Leu Leu Phe Ala Leu Met MetSer Leu Pro Ala Leu Phe 1770 1775 1780 1785 AAC ATC GGC CTC CTC CTC TTCCTC GTC ATG TTC ATC TAC TCC ATC TTC 5606 Asn Ile Gly Leu Leu Leu Phe LeuVal Met Phe Ile Tyr Ser Ile Phe 1790 1795 1800 GGC ATG GCC AGC TTC GCTAAC GTC GTG GAC GAG GCC GGC ATC GAC GAC 5654 Gly Met Ala Ser Phe Ala AsnVal Val Asp Glu Ala Gly Ile Asp Asp 1805 1810 1815 ATG TTC AAC TTC AAGACC TTT GGC AAC AGC ATG CTG TGC CTG TTC CAG 5702 Met Phe Asn Phe Lys ThrPhe Gly Asn Ser Met Leu Cys Leu Phe Gln 1820 1825 1830 ATC ACC ACC TCGGCC GGC TGG GAC GGC CTC CTC AGC CCC ATC CTC AAC 5750 Ile Thr Thr Ser AlaGly Trp Asp Gly Leu Leu Ser Pro Ile Leu Asn 1835 1840 1845 ACG GGG CCTCCC TAC TGC GAC CCC AAC CTG CCC AAC AGC AAC GGC TCC 5798 Thr Gly Pro ProTyr Cys Asp Pro Asn Leu Pro Asn Ser Asn Gly Ser 1850 1855 1860 1865 CGGGGG AAC TGC GGG AGC CCG GCG GTG GGC ATC ATC TTC TTC ACC ACC 5846 Arg GlyAsn Cys Gly Ser Pro Ala Val Gly Ile Ile Phe Phe Thr Thr 1870 1875 1880TAC ATC ATC ATC TCC TTC CTC ATC GTG GTC AAC ATG TAC ATC GCA GTG 5894 TyrIle Ile Ile Ser Phe Leu Ile Val Val Asn Met Tyr Ile Ala Val 1885 18901895 ATT CTG GAG AAC TTC AAC GTA GCC ACC GAG GAG AGC ACG GAG CCC CTG5942 Ile Leu Glu Asn Phe Asn Val Ala Thr Glu Glu Ser Thr Glu Pro Leu1900 1905 1910 AGC GAG GAC GAC TTC GAC ATG TTC TAT GAG ACC TGG GAG AAGTTC GAC 5990 Ser Glu Asp Asp Phe Asp Met Phe Tyr Glu Thr Trp Glu Lys PheAsp 1915 1920 1925 CCG GAG GCC ACC CAG TTC ATT GCC TTT TCT GCC CTC TCAGAC TTC GCG 6038 Pro Glu Ala Thr Gln Phe Ile Ala Phe Ser Ala Leu Ser AspPhe Ala 1930 1935 1940 1945 GAC ACG CTC TCC GGC CCT CTT AGA ATC CCC AAACCC AAC CAG AAT ATA 6086 Asp Thr Leu Ser Gly Pro Leu Arg Ile Pro Lys ProAsn Gln Asn Ile 1950 1955 1960 TTA ATC CAG ATG GAC CTG CCG TTG GTC CCCGGG GAT AAG ATC CAC TGT 6134 Leu Ile Gln Met Asp Leu Pro Leu Val Pro GlyAsp Lys Ile His Cys 1965 1970 1975 CTG GAC ATC CTT TTT GCC TTC ACA AAGAAC GTC TTG GGA GAA TCC GGG 6182 Leu Asp Ile Leu Phe Ala Phe Thr Lys AsnVal Leu Gly Glu Ser Gly 1980 1985 1990 GAG TTG GAC TCC CTG AAG ACC AATATG GAA GAG AAG TTT ATG GCG ACC 6230 Glu Leu Asp Ser Leu Lys Thr Asn MetGlu Glu Lys Phe Met Ala Thr 1995 2000 2005 AAT CTC TCC AAA GCA TCC TATGAA CCA ATA GCC ACC ACC CTC CGG TGG 6278 Asn Leu Ser Lys Ala Ser Tyr GluPro Ile Ala Thr Thr Leu Arg Trp 2010 2015 2020 2025 AAG CAG GAA GAC CTCTCA GCC ACA GTC ATT CAA AAG GCC TAC CGG AGC 6326 Lys Gln Glu Asp Leu SerAla Thr Val Ile Gln Lys Ala Tyr Arg Ser 2030 2035 2040 TAC ATG CTG CACCGC TCC TTG ACA CTC TCC AAC ACC CTG CAT GTG CCC 6374 Tyr Met Leu His ArgSer Leu Thr Leu Ser Asn Thr Leu His Val Pro 2045 2050 2055 AGG GCT GAGGAG GAT GGC GTG TCA CTT CCC GGG GAA GGC TAC AGT ACA 6422 Arg Ala Glu GluAsp Gly Val Ser Leu Pro Gly Glu Gly Tyr Ser Thr 2060 2065 2070 TTC ATGGCA AAC AGT GGA CTC CCG GAC AAA TCA GAA ACT GCC TCT GCT 6470 Phe Met AlaAsn Ser Gly Leu Pro Asp Lys Ser Glu Thr Ala Ser Ala 2075 2080 2085 ACGTCT TTC CCG CCA TCC TAT GAC AGT GTC ACC AGG GGC CTG AGT GAC 6518 Thr SerPhe Pro Pro Ser Tyr Asp Ser Val Thr Arg Gly Leu Ser Asp 2090 2095 21002105 CGG GCC AAC ATT AAC CCA TCT AGC TCA ATG CAA AAT GAA GAT GAG GTC6566 Arg Ala Asn Ile Asn Pro Ser Ser Ser Met Gln Asn Glu Asp Glu Val2110 2115 2120 GCT GCT AAG GAA GGA AAC AGC CCT GGA CCT CAG TGAAGGCACTCAGGCATGCA 6619 Ala Ala Lys Glu Gly Asn Ser Pro Gly Pro Gln 2125 2130CAGGGCAGGT TCCAATGTCT TTCTCTGCTG TACTAACTCC TTCCCTCTGG AGGTGGCACC 6679AACCTCCAGC CTCCACCAAT GCATGTCACT GGTCATGGTG TCAGAACTGA ATGGGGACAT 6739CCTTGAGAAA GCCCCCACCC CAATAGGAAT CAAAAGCCAA GGATACTCCT CCATTCTGAC 6799GTCCCTTCCG AGTTCCCAGA AGATGTCATT GCTCCCTTCT GTTTGTGACC AGAGACGTGA 6859TTCACCAACT TCTCGGAGCC AGAGACACAT AGCAAAGACT TTTCTGCTGG TGTCGGGCAG 6919TCTTAGAGAA GTCACGTAGG GGTTGGTACT GAGAATTAGG GTTTGCATGA CTGCATGCTC 6979ACAGCTGCCG GACAATACCT GTGAGTCGGC CATTAAAATT AATATTTTTA AAGTTAAAAA 7039AAAAAAAAAA AAA 7052 2132 amino acids amino acid linear protein 6 Met GluLeu Pro Phe Ala Ser Val Gly Thr Thr Asn Phe Arg Arg Phe 1 5 10 15 ThrPro Glu Ser Leu Ala Glu Ile Glu Lys Gln Ile Ala Ala His Arg 20 25 30 AlaAla Lys Lys Ala Arg Thr Lys His Arg Gly Gln Glu Asp Lys Gly 35 40 45 GluLys Pro Arg Pro Gln Leu Asp Leu Lys Asp Cys Asn Gln Leu Pro 50 55 60 LysPhe Tyr Gly Glu Leu Pro Ala Glu Leu Val Gly Glu Pro Leu Glu 65 70 75 80Asp Leu Asp Pro Phe Tyr Ser Thr His Arg Thr Phe Met Val Leu Asn 85 90 95Lys Ser Arg Thr Ile Ser Arg Phe Ser Ala Thr Trp Ala Leu Trp Leu 100 105110 Phe Ser Pro Phe Asn Leu Ile Arg Arg Thr Ala Ile Lys Val Ser Val 115120 125 His Ser Trp Phe Ser Ile Phe Ile Thr Ile Thr Ile Leu Val Asn Cys130 135 140 Val Cys Met Thr Arg Thr Asp Leu Pro Glu Lys Val Glu Tyr ValPhe 145 150 155 160 Thr Val Ile Tyr Thr Phe Glu Ala Leu Ile Lys Ile LeuAla Arg Gly 165 170 175 Phe Cys Leu Asn Glu Phe Thr Tyr Leu Arg Asp ProTrp Asn Trp Leu 180 185 190 Asp Phe Ser Val Ile Thr Leu Ala Tyr Val GlyAla Ala Ile Asp Leu 195 200 205 Arg Gly Ile Ser Gly Leu Arg Thr Phe ArgVal Leu Arg Ala Leu Lys 210 215 220 Thr Val Ser Val Ile Pro Gly Leu LysVal Ile Val Gly Ala Leu Ile 225 230 235 240 His Ser Val Arg Lys Leu AlaAsp Val Thr Ile Leu Thr Val Phe Cys 245 250 255 Leu Ser Val Phe Ala LeuVal Gly Leu Gln Leu Phe Lys Gly Asn Leu 260 265 270 Lys Asn Lys Cys IleArg Asn Gly Thr Asp Pro His Lys Ala Asp Asn 275 280 285 Leu Ser Ser GluMet Ala Glu Tyr Ile Phe Ile Lys Pro Gly Thr Thr 290 295 300 Asp Pro LeuLeu Cys Gly Asn Gly Ser Asp Ala Gly His Cys Pro Gly 305 310 315 320 GlyTyr Val Cys Leu Lys Thr Pro Asp Asn Pro Asp Phe Asn Tyr Thr 325 330 335Ser Phe Asp Ser Phe Ala Trp Ala Phe Leu Ser Leu Phe Arg Leu Met 340 345350 Thr Gln Asp Ser Trp Glu Arg Leu Tyr Gln Gln Thr Leu Arg Ala Ser 355360 365 Gly Lys Met Tyr Met Val Phe Phe Val Leu Val Ile Phe Leu Gly Ser370 375 380 Phe Tyr Leu Val Asn Leu Ile Leu Ala Val Val Thr Met Ala TyrGlu 385 390 395 400 Glu Gln Ser Gln Ala Thr Ile Ala Glu Ile Glu Ala LysGlu Lys Lys 405 410 415 Phe Gln Glu Ala Leu Glu Val Leu Gln Lys Glu GlnGlu Val Leu Ala 420 425 430 Ala Leu Gly Ile Asp Thr Thr Ser Leu Gln SerHis Ser Gly Ser Pro 435 440 445 Leu Ala Ser Lys Asn Ala Asn Glu Arg ArgPro Arg Val Lys Ser Arg 450 455 460 Val Ser Glu Gly Ser Thr Asp Asp AsnArg Ser Pro Gln Ser Asp Pro 465 470 475 480 Tyr Asn Gln Arg Arg Met SerPhe Leu Gly Leu Ser Ser Gly Arg Arg 485 490 495 Arg Ala Ser His Gly SerVal Phe His Phe Arg Ala Pro Ser Gln Asp 500 505 510 Ile Ser Phe Pro AspGly Ile Thr Pro Asp Asp Gly Val Phe His Gly 515 520 525 Asp Gln Glu SerArg Arg Gly Ser Ile Leu Leu Gly Arg Gly Ala Gly 530 535 540 Gln Thr GlyPro Leu Pro Arg Ser Pro Leu Pro Gln Ser Pro Asn Pro 545 550 555 560 GlyArg Arg His Gly Glu Glu Gly Gln Leu Gly Val Pro Thr Gly Glu 565 570 575Leu Thr Ala Gly Ala Pro Glu Gly Pro Ala Leu Asp Thr Thr Gly Gln 580 585590 Lys Ser Phe Leu Ser Ala Gly Tyr Leu Asn Glu Pro Phe Arg Ala Gln 595600 605 Arg Ala Met Ser Val Val Ser Ile Met Thr Ser Val Ile Glu Glu Leu610 615 620 Glu Glu Ser Lys Leu Lys Cys Pro Pro Cys Leu Ile Ser Phe AlaGln 625 630 635 640 Lys Tyr Leu Ile Trp Glu Cys Cys Pro Lys Trp Arg LysPhe Lys Met 645 650 655 Ala Leu Phe Glu Leu Val Thr Asp Pro Phe Ala GluLeu Thr Ile Thr 660 665 670 Leu Cys Ile Val Val Asn Thr Val Phe Met AlaMet Glu His Tyr Pro 675 680 685 Met Thr Asp Ala Phe Asp Ala Met Leu GlnAla Gly Asn Ile Val Phe 690 695 700 Thr Val Phe Phe Thr Met Glu Met AlaPhe Lys Ile Ile Ala Phe Asp 705 710 715 720 Pro Tyr Tyr Tyr Phe Gln LysLys Trp Asn Ile Phe Asp Cys Val Ile 725 730 735 Val Thr Val Ser Leu LeuGlu Leu Ser Ala Ser Lys Lys Gly Ser Leu 740 745 750 Ser Val Leu Arg SerLeu Arg Leu Ala Leu Asp Thr Thr Gly Gln Lys 755 760 765 Ser Phe Leu SerAla Gly Tyr Leu Asn Glu Pro Phe Arg Ala Gln Arg 770 775 780 Ala Met SerVal Val Ser Ile Met Thr Ser Val Ile Glu Glu Leu Glu 785 790 795 800 GluSer Lys Leu Lys Cys Pro Pro Cys Leu Ile Ser Phe Ala Gln Lys 805 810 815Tyr Leu Ile Trp Glu Cys Cys Pro Lys Trp Arg Lys Phe Lys Met Ala 820 825830 Leu Phe Glu Leu Val Thr Asp Pro Phe Ala Glu Leu Thr Ile Thr Leu 835840 845 Cys Ile Val Val Asn Thr Val Phe Met Ala Met Glu His Tyr Pro Met850 855 860 Thr Asp Ala Phe Asp Ala Met Leu Gln Ala Gly Asn Ile Val PheThr 865 870 875 880 Val Phe Phe Thr Met Glu Met Ala Phe Lys Ile Ile AlaPhe Asp Pro 885 890 895 Tyr Tyr Tyr Phe Gln Lys Lys Trp Asn Ile Phe AspCys Val Ile Val 900 905 910 Thr Val Ser Leu Leu Glu Leu Ser Ala Ser LysLys Gly Ser Leu Ser 915 920 925 Val Leu Arg Ser Leu Arg Leu Leu Arg ValPhe Lys Leu Ala Lys Ser 930 935 940 Trp Pro Thr Leu Asn Thr Leu Ile LysIle Ile Gly Asn Ser Val Gly 945 950 955 960 Ala Leu Gly Asn Leu Thr PheIle Leu Ala Ile Ile Val Phe Ile Phe 965 970 975 Ala Leu Val Gly Lys GlnLeu Leu Ser Glu Asp Tyr Gly Cys Arg Lys 980 985 990 Asp Gly Val Ser ValTrp Asn Gly Glu Lys Leu Arg Trp His Met Cys 995 1000 1005 Asp Phe PheHis Ser Phe Leu Val Val Phe Arg Ile Leu Cys Gly Glu 1010 1015 1020 TrpIle Glu Asn Met Trp Val Cys Met Glu Val Ser Gln Lys Ser Ile 1025 10301035 1040 Cys Leu Ile Leu Phe Leu Thr Val Met Val Leu Gly Asn Leu ValVal 1045 1050 1055 Leu Asn Leu Phe Ile Ala Leu Leu Leu Asn Ser Phe SerAla Asp Asn 1060 1065 1070 Leu Thr Ala Pro Glu Asp Asp Gly Glu Val AsnAsn Leu Gln Leu Ala 1075 1080 1085 Leu Ala Arg Ile Gln Val Leu Gly HisArg Ala Ser Arg Ala Ile Ala 1090 1095 1100 Ser Tyr Ile Ser Ser His CysArg Phe Arg Trp Pro Lys Val Glu Thr 1105 1110 1115 1120 Gln Leu Gly MetLys Pro Pro Leu Thr Ser Ser Glu Ala Lys Asn His 1125 1130 1135 Ile AlaThr Asp Ala Val Ser Ala Ala Val Gly Asn Leu Thr Lys Pro 1140 1145 1150Ala Leu Ser Ser Pro Lys Glu Asn His Gly Asp Phe Ile Thr Asp Pro 11551160 1165 Asn Val Trp Val Ser Val Pro Ile Ala Glu Gly Glu Ser Asp LeuAsp 1170 1175 1180 Glu Leu Glu Glu Asp Met Glu Gln Ala Ser Gln Ser SerTrp Gln Glu 1185 1190 1195 1200 Glu Asp Pro Lys Gly Gln Gln Glu Gln LeuPro Gln Val Gln Lys Cys 1205 1210 1215 Glu Asn His Gln Ala Ala Arg SerPro Ala Ser Met Met Ser Ser Glu 1220 1225 1230 Asp Leu Ala Pro Tyr LeuGly Glu Ser Trp Lys Arg Lys Asp Ser Pro 1235 1240 1245 Gln Val Pro AlaGlu Gly Val Asp Asp Thr Ser Ser Ser Glu Gly Ser 1250 1255 1260 Thr ValAsp Cys Pro Asp Pro Glu Glu Ile Leu Arg Lys Ile Pro Glu 1265 1270 12751280 Leu Ala Asp Asp Leu Asp Glu Pro Asp Asp Cys Phe Thr Glu Gly Cys1285 1290 1295 Thr Arg Arg Cys Pro Cys Cys Asn Val Asn Thr Ser Lys SerPro Trp 1300 1305 1310 Ala Thr Gly Trp Gln Val Arg Lys Thr Cys Tyr ArgIle Val Glu His 1315 1320 1325 Ser Trp Phe Glu Ser Phe Ile Ile Phe MetIle Leu Leu Ser Ser Gly 1330 1335 1340 Ala Leu Ala Phe Glu Asp Asn TyrLeu Glu Glu Lys Pro Arg Val Lys 1345 1350 1355 1360 Ser Val Leu Glu TyrThr Asp Arg Val Phe Thr Phe Ile Phe Val Phe 1365 1370 1375 Glu Met LeuLeu Lys Trp Val Ala Tyr Gly Phe Lys Lys Tyr Phe Thr 1380 1385 1390 AsnAla Trp Cys Trp Leu Asp Phe Leu Ile Val Asn Ile Ser Leu Thr 1395 14001405 Ser Leu Ile Ala Lys Ile Leu Glu Tyr Ser Asp Val Ala Ser Ile Lys1410 1415 1420 Ala Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala LeuSer Arg 1425 1430 1435 1440 Phe Glu Gly Met Arg Val Val Val Asp Ala LeuVal Gly Ala Ile Pro 1445 1450 1455 Ser Ile Met Asn Val Leu Leu Val CysLeu Ile Phe Trp Leu Ile Phe 1460 1465 1470 Ser Ile Met Gly Val Asn LeuPhe Ala Gly Lys Phe Ser Lys Cys Val 1475 1480 1485 Asp Thr Arg Asn AsnPro Phe Ser Asn Val Asn Ser Thr Met Val Asn 1490 1495 1500 Asn Lys SerGlu Cys His Asn Gln Asn Ser Thr Gly His Phe Phe Trp 1505 1510 1515 1520Val Asn Val Lys Val Asn Phe Asp Asn Val Ala Met Gly Tyr Leu Ala 15251530 1535 Leu Leu Gln Val Ala Thr Phe Lys Gly Trp Met Asp Ile Met TyrAla 1540 1545 1550 Ala Val Asp Ser Gly Glu Ile Asn Ser Gln Pro Asn TrpGlu Asn Asn 1555 1560 1565 Leu Tyr Met Tyr Leu Tyr Phe Val Val Phe IleIle Phe Gly Gly Phe 1570 1575 1580 Phe Thr Leu Asn Leu Phe Val Gly ValIle Ile Asp Asn Phe Asn Gln 1585 1590 1595 1600 Gln Lys Lys Lys Leu GlyGly Gln Asp Ile Phe Met Thr Glu Glu Gln 1605 1610 1615 Lys Lys Tyr TyrAsn Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln 1620 1625 1630 Lys ProIle Pro Arg Pro Leu Asn Lys Tyr Gln Gly Phe Val Phe Asp 1635 1640 1645Ile Val Thr Arg Gln Ala Phe Asp Ile Ile Ile Met Val Leu Ile Cys 16501655 1660 Leu Asn Met Ile Thr Met Met Val Glu Thr Asp Glu Gln Gly GluGlu 1665 1670 1675 1680 Lys Thr Lys Val Leu Gly Arg Ile Asn Gln Phe PheVal Ala Val Phe 1685 1690 1695 Thr Gly Glu Cys Val Met Lys Met Phe AlaLeu Arg Gln Tyr Tyr Phe 1700 1705 1710 Thr Asn Gly Trp Asn Val Phe AspPhe Ile Val Val Ile Leu Ser Ile 1715 1720 1725 Gly Ser Leu Leu Phe SerAla Ile Leu Lys Ser Leu Glu Asn Tyr Phe 1730 1735 1740 Ser Pro Thr LeuPhe Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile 1745 1750 1755 1760 LeuArg Leu Ile Arg Ala Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala 1765 17701775 Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe1780 1785 1790 Leu Val Met Phe Ile Tyr Ser Ile Phe Gly Met Ala Ser PheAla Asn 1795 1800 1805 Val Val Asp Glu Ala Gly Ile Asp Asp Met Phe AsnPhe Lys Thr Phe 1810 1815 1820 Gly Asn Ser Met Leu Cys Leu Phe Gln IleThr Thr Ser Ala Gly Trp 1825 1830 1835 1840 Asp Gly Leu Leu Ser Pro IleLeu Asn Thr Gly Pro Pro Tyr Cys Asp 1845 1850 1855 Pro Asn Leu Pro AsnSer Asn Gly Ser Arg Gly Asn Cys Gly Ser Pro 1860 1865 1870 Ala Val GlyIle Ile Phe Phe Thr Thr Tyr Ile Ile Ile Ser Phe Leu 1875 1880 1885 IleVal Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn Phe Asn Val 1890 18951900 Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp Phe Asp Met1905 1910 1915 1920 Phe Tyr Glu Thr Trp Glu Lys Phe Asp Pro Glu Ala ThrGln Phe Ile 1925 1930 1935 Ala Phe Ser Ala Leu Ser Asp Phe Ala Asp ThrLeu Ser Gly Pro Leu 1940 1945 1950 Arg Ile Pro Lys Pro Asn Gln Asn IleLeu Ile Gln Met Asp Leu Pro 1955 1960 1965 Leu Val Pro Gly Asp Lys IleHis Cys Leu Asp Ile Leu Phe Ala Phe 1970 1975 1980 Thr Lys Asn Val LeuGly Glu Ser Gly Glu Leu Asp Ser Leu Lys Thr 1985 1990 1995 2000 Asn MetGlu Glu Lys Phe Met Ala Thr Asn Leu Ser Lys Ala Ser Tyr 2005 2010 2015Glu Pro Ile Ala Thr Thr Leu Arg Trp Lys Gln Glu Asp Leu Ser Ala 20202025 2030 Thr Val Ile Gln Lys Ala Tyr Arg Ser Tyr Met Leu His Arg SerLeu 2035 2040 2045 Thr Leu Ser Asn Thr Leu His Val Pro Arg Ala Glu GluAsp Gly Val 2050 2055 2060 Ser Leu Pro Gly Glu Gly Tyr Ser Thr Phe MetAla Asn Ser Gly Leu 2065 2070 2075 2080 Pro Asp Lys Ser Glu Thr Ala SerAla Thr Ser Phe Pro Pro Ser Tyr 2085 2090 2095 Asp Ser Val Thr Arg GlyLeu Ser Asp Arg Ala Asn Ile Asn Pro Ser 2100 2105 2110 Ser Ser Met GlnAsn Glu Asp Glu Val Ala Ala Lys Glu Gly Asn Ser 2115 2120 2125 Pro GlyPro Gln 2130 6527 base pairs nucleic acid single linear cDNA CDS204..6077 7 TAGCTTGCTT CTGCTAATGC TACCCCAGGC CTTTAGACAG AGAACAGATGGCAGATGGAG 60 TTTCTTATTG CCATGCGCAA ACGCTGAGCC CACCTCATGA TCCCGGACCCCATGGTTTTC 120 AGTAGACAAC CTGGGCTAAG AAGAGATCTC CGACCTTATA GAGCAGCAAAGAGTGTAAAT 180 TCTTCCCCAA GAAGAATGAG AAG ATG GAG CTC CCC TTT GCG TCC GTGGGA 230 Met Glu Leu Pro Phe Ala Ser Val Gly 1 5 ACT ACC AAT TTC AGA CGGTTC ACT CCA GAG TCA CTG GCA GAG ATC GAG 278 Thr Thr Asn Phe Arg Arg PheThr Pro Glu Ser Leu Ala Glu Ile Glu 10 15 20 25 AAG CAG ATT GCT GCT CACCGG GCA GCC AAG AAG GCC AGA ACC AAG CAC 326 Lys Gln Ile Ala Ala His ArgAla Ala Lys Lys Ala Arg Thr Lys His 30 35 40 AGA GGA CAG GAG GAC AAG GGCGAG AAG CCC AGG CCT CAG CTG GAC TTG 374 Arg Gly Gln Glu Asp Lys Gly GluLys Pro Arg Pro Gln Leu Asp Leu 45 50 55 AAA GAC TGT AAC CAG CTG CCC AAGTTC TAT GGT GAG CTC CCA GCA GAA 422 Lys Asp Cys Asn Gln Leu Pro Lys PheTyr Gly Glu Leu Pro Ala Glu 60 65 70 CTG GTC GGG GAG CCC CTG GAG GAC CTAGAC CCT TTC TAC AGC ACA CAC 470 Leu Val Gly Glu Pro Leu Glu Asp Leu AspPro Phe Tyr Ser Thr His 75 80 85 CGG ACA TTC ATG GTG TTG AAT AAA AGC AGGACC ATT TCC AGA TTC AGT 518 Arg Thr Phe Met Val Leu Asn Lys Ser Arg ThrIle Ser Arg Phe Ser 90 95 100 105 GCC ACT TGG GCC CTG TGG CTC TTC AGTCCC TTC AAC CTG ATC AGA AGA 566 Ala Thr Trp Ala Leu Trp Leu Phe Ser ProPhe Asn Leu Ile Arg Arg 110 115 120 ACA GCC ATC AAA GTG TCT GTC CAT TCCTGG TTC TCC ATA TTC ATC ACC 614 Thr Ala Ile Lys Val Ser Val His Ser TrpPhe Ser Ile Phe Ile Thr 125 130 135 ATC ACT ATT TTG GTC AAC TGC GTG TGCATG ACC CGA ACT GAT CTT CCA 662 Ile Thr Ile Leu Val Asn Cys Val Cys MetThr Arg Thr Asp Leu Pro 140 145 150 GAG AAA GTC GAG TAC GTC TTC ACT GTCATT TAC ACC TTC GAG GCT CTG 710 Glu Lys Val Glu Tyr Val Phe Thr Val IleTyr Thr Phe Glu Ala Leu 155 160 165 ATT AAG ATA CTG GCA AGA GGG TTT TGTCTA AAT GAG TTC ACT TAT CTT 758 Ile Lys Ile Leu Ala Arg Gly Phe Cys LeuAsn Glu Phe Thr Tyr Leu 170 175 180 185 CGA GAT CCG TGG AAC TGG CTG GACTTC AGT GTC ATT ACC TTG GCG TAT 806 Arg Asp Pro Trp Asn Trp Leu Asp PheSer Val Ile Thr Leu Ala Tyr 190 195 200 GTG GGT GCA GCG ATA GAC CTC CGAGGA ATC TCA GGC CTG CGG ACA TTC 854 Val Gly Ala Ala Ile Asp Leu Arg GlyIle Ser Gly Leu Arg Thr Phe 205 210 215 CGA GTT CTC AGA GCC CTG AAA ACTGTT TCT GTG ATC CCA GGA CTG AAG 902 Arg Val Leu Arg Ala Leu Lys Thr ValSer Val Ile Pro Gly Leu Lys 220 225 230 GTC ATC GTG GGA GCC CTG ATC CACTCA GTG AGG AAG CTG GCC GAC GTG 950 Val Ile Val Gly Ala Leu Ile His SerVal Arg Lys Leu Ala Asp Val 235 240 245 ACT ATC CTC ACA GTC TTC TGC CTGAGC GTC TTC GCC TTG GTG GGC CTG 998 Thr Ile Leu Thr Val Phe Cys Leu SerVal Phe Ala Leu Val Gly Leu 250 255 260 265 CAG CTC TTT AAG GGG AAC CTTAAG AAC AAA TGC ATC AGG AAC GGA ACA 1046 Gln Leu Phe Lys Gly Asn Leu LysAsn Lys Cys Ile Arg Asn Gly Thr 270 275 280 GAT CCC CAC AAG GCT GAC AACCTC TCA TCT GAA ATG GCA GAA TAC ATC 1094 Asp Pro His Lys Ala Asp Asn LeuSer Ser Glu Met Ala Glu Tyr Ile 285 290 295 TTC ATC AAG CCT GGT ACT ACGGAT CCC TTA CTG TGC GGC AAT GGG TCT 1142 Phe Ile Lys Pro Gly Thr Thr AspPro Leu Leu Cys Gly Asn Gly Ser 300 305 310 GAT GCT GGT CAC TGC CCT GGAGGC TAT GTC TGC CTG AAA ACT CCT GAC 1190 Asp Ala Gly His Cys Pro Gly GlyTyr Val Cys Leu Lys Thr Pro Asp 315 320 325 AAC CCG GAT TTT AAC TAC ACCAGC TTT GAT TCC TTT GCG TGG GCA TTC 1238 Asn Pro Asp Phe Asn Tyr Thr SerPhe Asp Ser Phe Ala Trp Ala Phe 330 335 340 345 CTC TCA CTG TTC CGC CTCATG ACG CAG GAC TCC TGG GAG CGC CTG TAC 1286 Leu Ser Leu Phe Arg Leu MetThr Gln Asp Ser Trp Glu Arg Leu Tyr 350 355 360 CAG CAG ACA CTC CGG GCTTCT GGG AAA ATG TAC ATG GTC TTT TTC GTG 1334 Gln Gln Thr Leu Arg Ala SerGly Lys Met Tyr Met Val Phe Phe Val 365 370 375 CTG GTT ATT TTC CTT GGATCG TTC TAC CTG GTC AAT TTG ATC TTG GCC 1382 Leu Val Ile Phe Leu Gly SerPhe Tyr Leu Val Asn Leu Ile Leu Ala 380 385 390 GTG GTC ACC ATG GCG TATGAA GAG CAG AGC CAG GCA ACA ATT GCA GAA 1430 Val Val Thr Met Ala Tyr GluGlu Gln Ser Gln Ala Thr Ile Ala Glu 395 400 405 ATC GAA GCC AAG GAA AAAAAG TTC CAG GAA GCC CTT GAG GTG CTG CAG 1478 Ile Glu Ala Lys Glu Lys LysPhe Gln Glu Ala Leu Glu Val Leu Gln 410 415 420 425 AAG GAA CAG GAG GTGCTG GCA GCC CTG GGG ATT GAC ACG ACC TCG CTC 1526 Lys Glu Gln Glu Val LeuAla Ala Leu Gly Ile Asp Thr Thr Ser Leu 430 435 440 CAG TCC CAC AGT GGATCA CCC TTA GCC TCC AAA AAC GCC AAT GAG AGA 1574 Gln Ser His Ser Gly SerPro Leu Ala Ser Lys Asn Ala Asn Glu Arg 445 450 455 AGA CCC AGG GTG AAATCA AGG GTG TCA GAG GGC TCC ACG GAT GAC AAC 1622 Arg Pro Arg Val Lys SerArg Val Ser Glu Gly Ser Thr Asp Asp Asn 460 465 470 AGG TCA CCC CAA TCTGAC CCT TAC AAC CAG CGC AGG ATG TCT TTC CTA 1670 Arg Ser Pro Gln Ser AspPro Tyr Asn Gln Arg Arg Met Ser Phe Leu 475 480 485 GGC CTG TCT TCA GGAAGA CGC AGG GCT AGC CAC GGC AGT GTG TTC CAC 1718 Gly Leu Ser Ser Gly ArgArg Arg Ala Ser His Gly Ser Val Phe His 490 495 500 505 TTC CGA GCG CCCAGC CAA GAC ATC TCA TTT CCT GAC GGG ATC ACC CCT 1766 Phe Arg Ala Pro SerGln Asp Ile Ser Phe Pro Asp Gly Ile Thr Pro 510 515 520 GAT GAT GGG GTCTTT CAC GGA GAC CAG GAA AGC CGT CGA GGT TCC ATA 1814 Asp Asp Gly Val PheHis Gly Asp Gln Glu Ser Arg Arg Gly Ser Ile 525 530 535 TTG CTG GGC AGGGGT GCT GGG CAG ACA GGT CCA CTC CCC AGG AGC CCA 1862 Leu Leu Gly Arg GlyAla Gly Gln Thr Gly Pro Leu Pro Arg Ser Pro 540 545 550 CTG CCT CAG TCCCCC AAC CCT GGC CGT AGA CAT GGA GAA GAG GGA CAG 1910 Leu Pro Gln Ser ProAsn Pro Gly Arg Arg His Gly Glu Glu Gly Gln 555 560 565 CTC GGA GTG CCCACT GGT GAG CTT ACC GCT GGA GCG CCT GAA GGC CCG 1958 Leu Gly Val Pro ThrGly Glu Leu Thr Ala Gly Ala Pro Glu Gly Pro 570 575 580 585 GCA CTC GACACT ACA GGG CAG AAG AGC TTC CTG TCT GCG GGC TAC TTG 2006 Ala Leu Asp ThrThr Gly Gln Lys Ser Phe Leu Ser Ala Gly Tyr Leu 590 595 600 AAC GAA CCTTTC CGA GCA CAG AGG GCC ATG AGC GTT GTC AGT ATC ATG 2054 Asn Glu Pro PheArg Ala Gln Arg Ala Met Ser Val Val Ser Ile Met 605 610 615 ACT TCT GTCATT GAG GAG CTT GAA GAG TCT AAG CTG AAG TGC CCA CCC 2102 Thr Ser Val IleGlu Glu Leu Glu Glu Ser Lys Leu Lys Cys Pro Pro 620 625 630 TGC TTG ATCAGC TTC GCT CAG AAG TAT CTG ATC TGG GAG TGC TGC CCC 2150 Cys Leu Ile SerPhe Ala Gln Lys Tyr Leu Ile Trp Glu Cys Cys Pro 635 640 645 AAG TGG AGGAAG TTC AAG ATG GCG CTG TTC GAG CTG GTG ACT GAC CCC 2198 Lys Trp Arg LysPhe Lys Met Ala Leu Phe Glu Leu Val Thr Asp Pro 650 655 660 665 TTC GCAGAG CTT ACC ATC ACC CTC TGC ATC GTG GTG AAC ACC GTC TTC 2246 Phe Ala GluLeu Thr Ile Thr Leu Cys Ile Val Val Asn Thr Val Phe 670 675 680 ATG GCCATG GAG CAC TAC CCC ATG ACC GAT GCC TTC GAT GCC ATG CTT 2294 Met Ala MetGlu His Tyr Pro Met Thr Asp Ala Phe Asp Ala Met Leu 685 690 695 CAA GCCGGC AAC ATT GTC TTC ACC GTG TTT TTC ACA ATG GAG ATG GCC 2342 Gln Ala GlyAsn Ile Val Phe Thr Val Phe Phe Thr Met Glu Met Ala 700 705 710 TTC AAGATC ATT GCC TTC GAC CCC TAC TAT TAC TTC CAG AAG AAG TGG 2390 Phe Lys IleIle Ala Phe Asp Pro Tyr Tyr Tyr Phe Gln Lys Lys Trp 715 720 725 AAT ATCTTC GAC TGT GTC ATC GTC ACC GTG AGC CTT CTG GAG CTG AGT 2438 Asn Ile PheAsp Cys Val Ile Val Thr Val Ser Leu Leu Glu Leu Ser 730 735 740 745 GCATCC AAG AAG GGC AGC CTG TCT GTG CTC CGT TCC TTA CGC TTG CTG 2486 Ala SerLys Lys Gly Ser Leu Ser Val Leu Arg Ser Leu Arg Leu Leu 750 755 760 CGGGTC TTC AAG CTG GCC AAG TCC TGG CCC ACC CTG AAC ACC CTC ATC 2534 Arg ValPhe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Thr Leu Ile 765 770 775 AAGATC ATC GGG AAC TCA GTG GGG GCC CTG GGC AAC CTG ACC TTT ATC 2582 Lys IleIle Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Phe Ile 780 785 790 CTGGCC ATC ATC GTC TTC ATC TTC GCC CTG GTC GGA AAG CAG CTT CTC 2630 Leu AlaIle Ile Val Phe Ile Phe Ala Leu Val Gly Lys Gln Leu Leu 795 800 805 TCAGAG GAC TAC GGG TGC CGC AAG GAC GGC GTC TCC GTG TGG AAC GGC 2678 Ser GluAsp Tyr Gly Cys Arg Lys Asp Gly Val Ser Val Trp Asn Gly 810 815 820 825GAG AAG CTC CGC TGG CAC ATG TGT GAC TTC TTC CAT TCC TTC CTG GTC 2726 GluLys Leu Arg Trp His Met Cys Asp Phe Phe His Ser Phe Leu Val 830 835 840GTC TTC CGA ATC CTC TGC GGG GAG TGG ATC GAG AAC ATG TGG GTC TGC 2774 ValPhe Arg Ile Leu Cys Gly Glu Trp Ile Glu Asn Met Trp Val Cys 845 850 855ATG GAG GTC AGC CAG AAA TCC ATC TGC CTC ATC CTC TTC TTG ACT GTG 2822 MetGlu Val Ser Gln Lys Ser Ile Cys Leu Ile Leu Phe Leu Thr Val 860 865 870ATG GTG CTG GGC AAC CTA GTG GTG CTC AAC CTT TTC ATC GCT TTA CTG 2870 MetVal Leu Gly Asn Leu Val Val Leu Asn Leu Phe Ile Ala Leu Leu 875 880 885CTG AAC TCC TTC AGC GCG GAC AAC CTC ACG GCT CCA GAG GAT GAC GGG 2918 LeuAsn Ser Phe Ser Ala Asp Asn Leu Thr Ala Pro Glu Asp Asp Gly 890 895 900905 GAG GTG AAC AAC TTG CAG TTA GCA CTG GCC AGG ATC CAG GTA CTT GGC 2966Glu Val Asn Asn Leu Gln Leu Ala Leu Ala Arg Ile Gln Val Leu Gly 910 915920 CAT CGG GCC AGC AGG GCC ATC GCC AGT TAC ATC AGC AGC CAC TGC CGA 3014His Arg Ala Ser Arg Ala Ile Ala Ser Tyr Ile Ser Ser His Cys Arg 925 930935 TTC CGC TGG CCC AAG GTG GAG ACC CAG CTG GGC ATG AAG CCC CCA CTC 3062Phe Arg Trp Pro Lys Val Glu Thr Gln Leu Gly Met Lys Pro Pro Leu 940 945950 ACC AGC TCA GAG GCC AAG AAC CAC ATT GCC ACT GAT GCT GTC AGT GCT 3110Thr Ser Ser Glu Ala Lys Asn His Ile Ala Thr Asp Ala Val Ser Ala 955 960965 GCA GTG GGG AAC CTG ACA AAG CCA GCT CTC AGT AGC CCC AAG GAG AAT 3158Ala Val Gly Asn Leu Thr Lys Pro Ala Leu Ser Ser Pro Lys Glu Asn 970 975980 985 CAC GGG GAC TTC ATC ACT GAT CCC AAC GTG TGG GTC TCT GTG CCC ATT3206 His Gly Asp Phe Ile Thr Asp Pro Asn Val Trp Val Ser Val Pro Ile 990995 1000 GCT GAG GGG GAA TCT GAC CTC GAC GAG CTC GAG GAA GAT ATG GAG CAG3254 Ala Glu Gly Glu Ser Asp Leu Asp Glu Leu Glu Glu Asp Met Glu Gln1005 1010 1015 GCT TCG CAG AGC TCC TGG CAG GAA GAG GAC CCC AAG GGA CAGCAG GAG 3302 Ala Ser Gln Ser Ser Trp Gln Glu Glu Asp Pro Lys Gly Gln GlnGlu 1020 1025 1030 CAG TTG CCA CAA GTC CAA AAG TGT GAA AAC CAC CAG GCAGCC AGA AGC 3350 Gln Leu Pro Gln Val Gln Lys Cys Glu Asn His Gln Ala AlaArg Ser 1035 1040 1045 CCA GCC TCC ATG ATG TCC TCT GAG GAC CTG GCT CCATAC CTG GGT GAG 3398 Pro Ala Ser Met Met Ser Ser Glu Asp Leu Ala Pro TyrLeu Gly Glu 1050 1055 1060 1065 AGC TGG AAG AGG AAG GAT AGC CCT CAG GTCCCT GCC GAG GGA GTG GAT 3446 Ser Trp Lys Arg Lys Asp Ser Pro Gln Val ProAla Glu Gly Val Asp 1070 1075 1080 GAC ACG AGC TCC TCT GAG GGC AGC ACGGTG GAC TGC CCG GAC CCA GAG 3494 Asp Thr Ser Ser Ser Glu Gly Ser Thr ValAsp Cys Pro Asp Pro Glu 1085 1090 1095 GAA ATC CTG AGG AAG ATC CCC GAGCTG GCA GAT GAC CTG GAC GAG CCC 3542 Glu Ile Leu Arg Lys Ile Pro Glu LeuAla Asp Asp Leu Asp Glu Pro 1100 1105 1110 GAT GAC TGT TTC ACA GAA GGCTGC ACT CGC CGC TGT CCC TGC TGC AAC 3590 Asp Asp Cys Phe Thr Glu Gly CysThr Arg Arg Cys Pro Cys Cys Asn 1115 1120 1125 GTG AAT ACT AGC AAG TCTCCT TGG GCC ACA GGC TGG CAG GTG CGC AAG 3638 Val Asn Thr Ser Lys Ser ProTrp Ala Thr Gly Trp Gln Val Arg Lys 1130 1135 1140 1145 ACC TGC TAC CGCATC GTG GAG CAC AGC TGG TTT GAG AGT TTC ATC ATC 3686 Thr Cys Tyr Arg IleVal Glu His Ser Trp Phe Glu Ser Phe Ile Ile 1150 1155 1160 TTC ATG ATCCTG CTC AGC AGT GGA GCG CTG GCC TTT GAG GAT AAC TAC 3734 Phe Met Ile LeuLeu Ser Ser Gly Ala Leu Ala Phe Glu Asp Asn Tyr 1165 1170 1175 CTG GAAGAG AAA CCC CGA GTG AAG TCC GTG CTG GAG TAC ACT GAC CGA 3782 Leu Glu GluLys Pro Arg Val Lys Ser Val Leu Glu Tyr Thr Asp Arg 1180 1185 1190 GTGTTC ACC TTC ATC TTC GTC TTT GAG ATG CTG CTC AAG TGG GTA GCC 3830 Val PheThr Phe Ile Phe Val Phe Glu Met Leu Leu Lys Trp Val Ala 1195 1200 1205TAT GGC TTC AAA AAG TAT TTC ACC AAT GCC TGG TGC TGG CTG GAC TTC 3878 TyrGly Phe Lys Lys Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe 1210 12151220 1225 CTC ATT GTG AAC ATC TCC CTG ACA AGC CTC ATA GCG AAG ATC CTTGAG 3926 Leu Ile Val Asn Ile Ser Leu Thr Ser Leu Ile Ala Lys Ile Leu Glu1230 1235 1240 TAT TCC GAC GTG GCG TCC ATC AAA GCC CTT CGG ACT CTC CGTGCC CTC 3974 Tyr Ser Asp Val Ala Ser Ile Lys Ala Leu Arg Thr Leu Arg AlaLeu 1245 1250 1255 CGA CCG CTG CGG GCT CTG TCT CGA TTC GAA GGC ATG AGGGTA GTG GTG 4022 Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg ValVal Val 1260 1265 1270 GAT GCC CTC GTG GGC GCC ATC CCC TCC ATC ATG AACGTC CTC CTC GTC 4070 Asp Ala Leu Val Gly Ala Ile Pro Ser Ile Met Asn ValLeu Leu Val 1275 1280 1285 TGC CTC ATC TTC TGG CTC ATC TTC AGC ATC ATGGGC GTG AAC CTC TTC 4118 Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met GlyVal Asn Leu Phe 1290 1295 1300 1305 GCC GGG AAA TTT TCG AAG TGC GTC GACACC AGA AAT AAC CCA TTT TCC 4166 Ala Gly Lys Phe Ser Lys Cys Val Asp ThrArg Asn Asn Pro Phe Ser 1310 1315 1320 AAC GTG AAT TCG ACG ATG GTG AATAAC AAG TCC GAG TGT CAC AAT CAA 4214 Asn Val Asn Ser Thr Met Val Asn AsnLys Ser Glu Cys His Asn Gln 1325 1330 1335 AAC AGC ACC GGC CAC TTC TTCTGG GTC AAC GTC AAA GTC AAC TTC GAC 4262 Asn Ser Thr Gly His Phe Phe TrpVal Asn Val Lys Val Asn Phe Asp 1340 1345 1350 AAC GTC GCT ATG GGC TACCTC GCA CTT CTT CAG GTG GCA ACC TTC AAA 4310 Asn Val Ala Met Gly Tyr LeuAla Leu Leu Gln Val Ala Thr Phe Lys 1355 1360 1365 GGC TGG ATG GAC ATAATG TAT GCA GCT GTT GAT TCC GGA GAG ATC AAC 4358 Gly Trp Met Asp Ile MetTyr Ala Ala Val Asp Ser Gly Glu Ile Asn 1370 1375 1380 1385 AGT CAG CCTAAC TGG GAG AAC AAC TTG TAC ATG TAC CTG TAC TTC GTC 4406 Ser Gln Pro AsnTrp Glu Asn Asn Leu Tyr Met Tyr Leu Tyr Phe Val 1390 1395 1400 GTT TTCATC ATT TTC GGT GGC TTC TTC ACG CTG AAT CTC TTT GTT GGG 4454 Val Phe IleIle Phe Gly Gly Phe Phe Thr Leu Asn Leu Phe Val Gly 1405 1410 1415 GTCATA ATC GAC AAC TTC AAC CAA CAG AAA AAA AAG CTA GGA GGC CAG 4502 Val IleIle Asp Asn Phe Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln 1420 1425 1430GAC ATC TTC ATG ACA GAA GAG CAG AAG AAG TAC TAC AAT GCC ATG AAG 4550 AspIle Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys 1435 14401445 AAG CTG GGC TCC AAG AAA CCC CAG AAG CCC ATC CCA CGG CCC CTG AAT4598 Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Leu Asn1450 1455 1460 1465 AAG TAC CAA GGC TTC GTG TTT GAC ATC GTG ACC AGG CAAGCC TTT GAC 4646 Lys Tyr Gln Gly Phe Val Phe Asp Ile Val Thr Arg Gln AlaPhe Asp 1470 1475 1480 ATC ATC ATC ATG GTT CTC ATC TGC CTC AAC ATG ATCACC ATG ATG GTG 4694 Ile Ile Ile Met Val Leu Ile Cys Leu Asn Met Ile ThrMet Met Val 1485 1490 1495 GAG ACC GAC GAG CAG GGC GAG GAG AAG ACG AAGGTT CTG GGC AGA ATC 4742 Glu Thr Asp Glu Gln Gly Glu Glu Lys Thr Lys ValLeu Gly Arg Ile 1500 1505 1510 AAC CAG TTC TTT GTG GCC GTC TTC ACG GGCGAG TGT GTG ATG AAG ATG 4790 Asn Gln Phe Phe Val Ala Val Phe Thr Gly GluCys Val Met Lys Met 1515 1520 1525 TTC GCC CTG CGA CAG TAC TAC TTC ACCAAC GGC TGG AAC GTG TTC GAC 4838 Phe Ala Leu Arg Gln Tyr Tyr Phe Thr AsnGly Trp Asn Val Phe Asp 1530 1535 1540 1545 TTC ATA GTG GTG ATC CTG TCCATT GGG AGT CTG CTG TTT TCT GCA ATC 4886 Phe Ile Val Val Ile Leu Ser IleGly Ser Leu Leu Phe Ser Ala Ile 1550 1555 1560 CTT AAG TCA CTG GAA AACTAC TTC TCC CCG ACG CTC TTC CGG GTC ATC 4934 Leu Lys Ser Leu Glu Asn TyrPhe Ser Pro Thr Leu Phe Arg Val Ile 1565 1570 1575 CGT CTG GCC AGG ATCGGC CGC ATC CTC AGG CTG ATC CGA GCA GCC AAG 4982 Arg Leu Ala Arg Ile GlyArg Ile Leu Arg Leu Ile Arg Ala Ala Lys 1580 1585 1590 GGG ATT CGC ACGCTG CTC TTC GCC CTC ATG ATG TCC CTG CCC GCC CTC 5030 Gly Ile Arg Thr LeuLeu Phe Ala Leu Met Met Ser Leu Pro Ala Leu 1595 1600 1605 TTC AAC ATCGGC CTC CTC CTC TTC CTC GTC ATG TTC ATC TAC TCC ATC 5078 Phe Asn Ile GlyLeu Leu Leu Phe Leu Val Met Phe Ile Tyr Ser Ile 1610 1615 1620 1625 TTCGGC ATG GCC AGC TTC GCT AAC GTC GTG GAC GAG GCC GGC ATC GAC 5126 Phe GlyMet Ala Ser Phe Ala Asn Val Val Asp Glu Ala Gly Ile Asp 1630 1635 1640GAC ATG TTC AAC TTC AAG ACC TTT GGC AAC AGC ATG CTG TGC CTG TTC 5174 AspMet Phe Asn Phe Lys Thr Phe Gly Asn Ser Met Leu Cys Leu Phe 1645 16501655 CAG ATC ACC ACC TCG GCC GGC TGG GAC GGC CTC CTC AGC CCC ATC CTC5222 Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Ser Pro Ile Leu1660 1665 1670 AAC ACG GGG CCT CCC TAC TGC GAC CCC AAC CTG CCC AAC AGCAAC GGC 5270 Asn Thr Gly Pro Pro Tyr Cys Asp Pro Asn Leu Pro Asn Ser AsnGly 1675 1680 1685 TCC CGG GGG AAC TGC GGG AGC CCG GCG GTG GGC ATC ATCTTC TTC ACC 5318 Ser Arg Gly Asn Cys Gly Ser Pro Ala Val Gly Ile Ile PhePhe Thr 1690 1695 1700 1705 ACC TAC ATC ATC ATC TCC TTC CTC ATC GTG GTCAAC ATG TAC ATC GCA 5366 Thr Tyr Ile Ile Ile Ser Phe Leu Ile Val Val AsnMet Tyr Ile Ala 1710 1715 1720 GTG ATT CTG GAG AAC TTC AAC GTA GCC ACCGAG GAG AGC ACG GAG CCC 5414 Val Ile Leu Glu Asn Phe Asn Val Ala Thr GluGlu Ser Thr Glu Pro 1725 1730 1735 CTG AGC GAG GAC GAC TTC GAC ATG TTCTAT GAG ACC TGG GAG AAG TTC 5462 Leu Ser Glu Asp Asp Phe Asp Met Phe TyrGlu Thr Trp Glu Lys Phe 1740 1745 1750 GAC CCG GAG GCC ACC CAG TTC ATTGCC TTT TCT GCC CTC TCA GAC TTC 5510 Asp Pro Glu Ala Thr Gln Phe Ile AlaPhe Ser Ala Leu Ser Asp Phe 1755 1760 1765 GCG GAC ACG CTC TCC GGC CCTCTT AGA ATC CCC AAA CCC AAC CAG AAT 5558 Ala Asp Thr Leu Ser Gly Pro LeuArg Ile Pro Lys Pro Asn Gln Asn 1770 1775 1780 1785 ATA TTA ATC CAG ATGGAC CTG CCG TTG GTC CCC GGG GAT AAG ATC CAC 5606 Ile Leu Ile Gln Met AspLeu Pro Leu Val Pro Gly Asp Lys Ile His 1790 1795 1800 TGT CTG GAC ATCCTT TTT GCC TTC ACA AAG AAC GTC TTG GGA GAA TCC 5654 Cys Leu Asp Ile LeuPhe Ala Phe Thr Lys Asn Val Leu Gly Glu Ser 1805 1810 1815 GGG GAG TTGGAC TCC CTG AAG ACC AAT ATG GAA GAG AAG TTT ATG GCG 5702 Gly Glu Leu AspSer Leu Lys Thr Asn Met Glu Glu Lys Phe Met Ala 1820 1825 1830 ACC AATCTC TCC AAA GCA TCC TAT GAA CCA ATA GCC ACC ACC CTC CGG 5750 Thr Asn LeuSer Lys Ala Ser Tyr Glu Pro Ile Ala Thr Thr Leu Arg 1835 1840 1845 TGGAAG CAG GAA GAC CTC TCA GCC ACA GTC ATT CAA AAG GCC TAC CGG 5798 Trp LysGln Glu Asp Leu Ser Ala Thr Val Ile Gln Lys Ala Tyr Arg 1850 1855 18601865 AGC TAC ATG CTG CAC CGC TCC TTG ACA CTC TCC AAC ACC CTG CAT GTG5846 Ser Tyr Met Leu His Arg Ser Leu Thr Leu Ser Asn Thr Leu His Val1870 1875 1880 CCC AGG GCT GAG GAG GAT GGC GTG TCA CTT CCC GGG GAA GGCTAC AGT 5894 Pro Arg Ala Glu Glu Asp Gly Val Ser Leu Pro Gly Glu Gly TyrSer 1885 1890 1895 ACA TTC ATG GCA AAC AGT GGA CTC CCG GAC AAA TCA GAAACT GCC TCT 5942 Thr Phe Met Ala Asn Ser Gly Leu Pro Asp Lys Ser Glu ThrAla Ser 1900 1905 1910 GCT ACG TCT TTC CCG CCA TCC TAT GAC AGT GTC ACCAGG GGC CTG AGT 5990 Ala Thr Ser Phe Pro Pro Ser Tyr Asp Ser Val Thr ArgGly Leu Ser 1915 1920 1925 GAC CGG GCC AAC ATT AAC CCA TCT AGC TCA ATGCAA AAT GAA GAT GAG 6038 Asp Arg Ala Asn Ile Asn Pro Ser Ser Ser Met GlnAsn Glu Asp Glu 1930 1935 1940 1945 GTC GCT GCT AAG GAA GGA AAC AGC CCTGGA CCT CAG TGAAGGCACT 6084 Val Ala Ala Lys Glu Gly Asn Ser Pro Gly ProGln 1950 1955 CAGGCATGCA CAGGGCAGGT TCCAATGTCT TTCTCTGCTG TACTAACTCCTTCCCTCTGG 6144 AGGTGGCACC AACCTCCAGC CTCCACCAAT GCATGTCACT GGTCATGGTGTCAGAACTGA 6204 ATGGGGACAT CCTTGAGAAA GCCCCCACCC CAATAGGAAT CAAAAGCCAAGGATACTCCT 6264 CCATTCTGAC GTCCCTTCCG AGTTCCCAGA AGATGTCATT GCTCCCTTCTGTTTGTGACC 6324 AGAGACGTGA TTCACCAACT TCTCGGAGCC AGAGACACAT AGCAAAGACTTTTCTGCTGG 6384 TGTCGGGCAG TCTTAGAGAA GTCACGTAGG GGTTGGTACT GAGAATTAGGGTTTGCATGA 6444 CTGCATGCTC ACAGCTGCCG GACAATACCT GTGAGTCGGC CATTAAAATTAATATTTTTA 6504 AAGTTAAAAA AAAAAAAAAA AAA 6527 1957 amino acids aminoacid linear protein 8 Met Glu Leu Pro Phe Ala Ser Val Gly Thr Thr AsnPhe Arg Arg Phe 1 5 10 15 Thr Pro Glu Ser Leu Ala Glu Ile Glu Lys GlnIle Ala Ala His Arg 20 25 30 Ala Ala Lys Lys Ala Arg Thr Lys His Arg GlyGln Glu Asp Lys Gly 35 40 45 Glu Lys Pro Arg Pro Gln Leu Asp Leu Lys AspCys Asn Gln Leu Pro 50 55 60 Lys Phe Tyr Gly Glu Leu Pro Ala Glu Leu ValGly Glu Pro Leu Glu 65 70 75 80 Asp Leu Asp Pro Phe Tyr Ser Thr His ArgThr Phe Met Val Leu Asn 85 90 95 Lys Ser Arg Thr Ile Ser Arg Phe Ser AlaThr Trp Ala Leu Trp Leu 100 105 110 Phe Ser Pro Phe Asn Leu Ile Arg ArgThr Ala Ile Lys Val Ser Val 115 120 125 His Ser Trp Phe Ser Ile Phe IleThr Ile Thr Ile Leu Val Asn Cys 130 135 140 Val Cys Met Thr Arg Thr AspLeu Pro Glu Lys Val Glu Tyr Val Phe 145 150 155 160 Thr Val Ile Tyr ThrPhe Glu Ala Leu Ile Lys Ile Leu Ala Arg Gly 165 170 175 Phe Cys Leu AsnGlu Phe Thr Tyr Leu Arg Asp Pro Trp Asn Trp Leu 180 185 190 Asp Phe SerVal Ile Thr Leu Ala Tyr Val Gly Ala Ala Ile Asp Leu 195 200 205 Arg GlyIle Ser Gly Leu Arg Thr Phe Arg Val Leu Arg Ala Leu Lys 210 215 220 ThrVal Ser Val Ile Pro Gly Leu Lys Val Ile Val Gly Ala Leu Ile 225 230 235240 His Ser Val Arg Lys Leu Ala Asp Val Thr Ile Leu Thr Val Phe Cys 245250 255 Leu Ser Val Phe Ala Leu Val Gly Leu Gln Leu Phe Lys Gly Asn Leu260 265 270 Lys Asn Lys Cys Ile Arg Asn Gly Thr Asp Pro His Lys Ala AspAsn 275 280 285 Leu Ser Ser Glu Met Ala Glu Tyr Ile Phe Ile Lys Pro GlyThr Thr 290 295 300 Asp Pro Leu Leu Cys Gly Asn Gly Ser Asp Ala Gly HisCys Pro Gly 305 310 315 320 Gly Tyr Val Cys Leu Lys Thr Pro Asp Asn ProAsp Phe Asn Tyr Thr 325 330 335 Ser Phe Asp Ser Phe Ala Trp Ala Phe LeuSer Leu Phe Arg Leu Met 340 345 350 Thr Gln Asp Ser Trp Glu Arg Leu TyrGln Gln Thr Leu Arg Ala Ser 355 360 365 Gly Lys Met Tyr Met Val Phe PheVal Leu Val Ile Phe Leu Gly Ser 370 375 380 Phe Tyr Leu Val Asn Leu IleLeu Ala Val Val Thr Met Ala Tyr Glu 385 390 395 400 Glu Gln Ser Gln AlaThr Ile Ala Glu Ile Glu Ala Lys Glu Lys Lys 405 410 415 Phe Gln Glu AlaLeu Glu Val Leu Gln Lys Glu Gln Glu Val Leu Ala 420 425 430 Ala Leu GlyIle Asp Thr Thr Ser Leu Gln Ser His Ser Gly Ser Pro 435 440 445 Leu AlaSer Lys Asn Ala Asn Glu Arg Arg Pro Arg Val Lys Ser Arg 450 455 460 ValSer Glu Gly Ser Thr Asp Asp Asn Arg Ser Pro Gln Ser Asp Pro 465 470 475480 Tyr Asn Gln Arg Arg Met Ser Phe Leu Gly Leu Ser Ser Gly Arg Arg 485490 495 Arg Ala Ser His Gly Ser Val Phe His Phe Arg Ala Pro Ser Gln Asp500 505 510 Ile Ser Phe Pro Asp Gly Ile Thr Pro Asp Asp Gly Val Phe HisGly 515 520 525 Asp Gln Glu Ser Arg Arg Gly Ser Ile Leu Leu Gly Arg GlyAla Gly 530 535 540 Gln Thr Gly Pro Leu Pro Arg Ser Pro Leu Pro Gln SerPro Asn Pro 545 550 555 560 Gly Arg Arg His Gly Glu Glu Gly Gln Leu GlyVal Pro Thr Gly Glu 565 570 575 Leu Thr Ala Gly Ala Pro Glu Gly Pro AlaLeu Asp Thr Thr Gly Gln 580 585 590 Lys Ser Phe Leu Ser Ala Gly Tyr LeuAsn Glu Pro Phe Arg Ala Gln 595 600 605 Arg Ala Met Ser Val Val Ser IleMet Thr Ser Val Ile Glu Glu Leu 610 615 620 Glu Glu Ser Lys Leu Lys CysPro Pro Cys Leu Ile Ser Phe Ala Gln 625 630 635 640 Lys Tyr Leu Ile TrpGlu Cys Cys Pro Lys Trp Arg Lys Phe Lys Met 645 650 655 Ala Leu Phe GluLeu Val Thr Asp Pro Phe Ala Glu Leu Thr Ile Thr 660 665 670 Leu Cys IleVal Val Asn Thr Val Phe Met Ala Met Glu His Tyr Pro 675 680 685 Met ThrAsp Ala Phe Asp Ala Met Leu Gln Ala Gly Asn Ile Val Phe 690 695 700 ThrVal Phe Phe Thr Met Glu Met Ala Phe Lys Ile Ile Ala Phe Asp 705 710 715720 Pro Tyr Tyr Tyr Phe Gln Lys Lys Trp Asn Ile Phe Asp Cys Val Ile 725730 735 Val Thr Val Ser Leu Leu Glu Leu Ser Ala Ser Lys Lys Gly Ser Leu740 745 750 Ser Val Leu Arg Ser Leu Arg Leu Leu Arg Val Phe Lys Leu AlaLys 755 760 765 Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys Ile Ile Gly AsnSer Val 770 775 780 Gly Ala Leu Gly Asn Leu Thr Phe Ile Leu Ala Ile IleVal Phe Ile 785 790 795 800 Phe Ala Leu Val Gly Lys Gln Leu Leu Ser GluAsp Tyr Gly Cys Arg 805 810 815 Lys Asp Gly Val Ser Val Trp Asn Gly GluLys Leu Arg Trp His Met 820 825 830 Cys Asp Phe Phe His Ser Phe Leu ValVal Phe Arg Ile Leu Cys Gly 835 840 845 Glu Trp Ile Glu Asn Met Trp ValCys Met Glu Val Ser Gln Lys Ser 850 855 860 Ile Cys Leu Ile Leu Phe LeuThr Val Met Val Leu Gly Asn Leu Val 865 870 875 880 Val Leu Asn Leu PheIle Ala Leu Leu Leu Asn Ser Phe Ser Ala Asp 885 890 895 Asn Leu Thr AlaPro Glu Asp Asp Gly Glu Val Asn Asn Leu Gln Leu 900 905 910 Ala Leu AlaArg Ile Gln Val Leu Gly His Arg Ala Ser Arg Ala Ile 915 920 925 Ala SerTyr Ile Ser Ser His Cys Arg Phe Arg Trp Pro Lys Val Glu 930 935 940 ThrGln Leu Gly Met Lys Pro Pro Leu Thr Ser Ser Glu Ala Lys Asn 945 950 955960 His Ile Ala Thr Asp Ala Val Ser Ala Ala Val Gly Asn Leu Thr Lys 965970 975 Pro Ala Leu Ser Ser Pro Lys Glu Asn His Gly Asp Phe Ile Thr Asp980 985 990 Pro Asn Val Trp Val Ser Val Pro Ile Ala Glu Gly Glu Ser AspLeu 995 1000 1005 Asp Glu Leu Glu Glu Asp Met Glu Gln Ala Ser Gln SerSer Trp Gln 1010 1015 1020 Glu Glu Asp Pro Lys Gly Gln Gln Glu Gln LeuPro Gln Val Gln Lys 1025 1030 1035 1040 Cys Glu Asn His Gln Ala Ala ArgSer Pro Ala Ser Met Met Ser Ser 1045 1050 1055 Glu Asp Leu Ala Pro TyrLeu Gly Glu Ser Trp Lys Arg Lys Asp Ser 1060 1065 1070 Pro Gln Val ProAla Glu Gly Val Asp Asp Thr Ser Ser Ser Glu Gly 1075 1080 1085 Ser ThrVal Asp Cys Pro Asp Pro Glu Glu Ile Leu Arg Lys Ile Pro 1090 1095 1100Glu Leu Ala Asp Asp Leu Asp Glu Pro Asp Asp Cys Phe Thr Glu Gly 11051110 1115 1120 Cys Thr Arg Arg Cys Pro Cys Cys Asn Val Asn Thr Ser LysSer Pro 1125 1130 1135 Trp Ala Thr Gly Trp Gln Val Arg Lys Thr Cys TyrArg Ile Val Glu 1140 1145 1150 His Ser Trp Phe Glu Ser Phe Ile Ile PheMet Ile Leu Leu Ser Ser 1155 1160 1165 Gly Ala Leu Ala Phe Glu Asp AsnTyr Leu Glu Glu Lys Pro Arg Val 1170 1175 1180 Lys Ser Val Leu Glu TyrThr Asp Arg Val Phe Thr Phe Ile Phe Val 1185 1190 1195 1200 Phe Glu MetLeu Leu Lys Trp Val Ala Tyr Gly Phe Lys Lys Tyr Phe 1205 1210 1215 ThrAsn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val Asn Ile Ser Leu 1220 12251230 Thr Ser Leu Ile Ala Lys Ile Leu Glu Tyr Ser Asp Val Ala Ser Ile1235 1240 1245 Lys Ala Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg AlaLeu Ser 1250 1255 1260 Arg Phe Glu Gly Met Arg Val Val Val Asp Ala LeuVal Gly Ala Ile 1265 1270 1275 1280 Pro Ser Ile Met Asn Val Leu Leu ValCys Leu Ile Phe Trp Leu Ile 1285 1290 1295 Phe Ser Ile Met Gly Val AsnLeu Phe Ala Gly Lys Phe Ser Lys Cys 1300 1305 1310 Val Asp Thr Arg AsnAsn Pro Phe Ser Asn Val Asn Ser Thr Met Val 1315 1320 1325 Asn Asn LysSer Glu Cys His Asn Gln Asn Ser Thr Gly His Phe Phe 1330 1335 1340 TrpVal Asn Val Lys Val Asn Phe Asp Asn Val Ala Met Gly Tyr Leu 1345 13501355 1360 Ala Leu Leu Gln Val Ala Thr Phe Lys Gly Trp Met Asp Ile MetTyr 1365 1370 1375 Ala Ala Val Asp Ser Gly Glu Ile Asn Ser Gln Pro AsnTrp Glu Asn 1380 1385 1390 Asn Leu Tyr Met Tyr Leu Tyr Phe Val Val PheIle Ile Phe Gly Gly 1395 1400 1405 Phe Phe Thr Leu Asn Leu Phe Val GlyVal Ile Ile Asp Asn Phe Asn 1410 1415 1420 Gln Gln Lys Lys Lys Leu GlyGly Gln Asp Ile Phe Met Thr Glu Glu 1425 1430 1435 1440 Gln Lys Lys TyrTyr Asn Ala Met Lys Lys Leu Gly Ser Lys Lys Pro 1445 1450 1455 Gln LysPro Ile Pro Arg Pro Leu Asn Lys Tyr Gln Gly Phe Val Phe 1460 1465 1470Asp Ile Val Thr Arg Gln Ala Phe Asp Ile Ile Ile Met Val Leu Ile 14751480 1485 Cys Leu Asn Met Ile Thr Met Met Val Glu Thr Asp Glu Gln GlyGlu 1490 1495 1500 Glu Lys Thr Lys Val Leu Gly Arg Ile Asn Gln Phe PheVal Ala Val 1505 1510 1515 1520 Phe Thr Gly Glu Cys Val Met Lys Met PheAla Leu Arg Gln Tyr Tyr 1525 1530 1535 Phe Thr Asn Gly Trp Asn Val PheAsp Phe Ile Val Val Ile Leu Ser 1540 1545 1550 Ile Gly Ser Leu Leu PheSer Ala Ile Leu Lys Ser Leu Glu Asn Tyr 1555 1560 1565 Phe Ser Pro ThrLeu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly Arg 1570 1575 1580 Ile LeuArg Leu Ile Arg Ala Ala Lys Gly Ile Arg Thr Leu Leu Phe 1585 1590 15951600 Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu1605 1610 1615 Phe Leu Val Met Phe Ile Tyr Ser Ile Phe Gly Met Ala SerPhe Ala 1620 1625 1630 Asn Val Val Asp Glu Ala Gly Ile Asp Asp Met PheAsn Phe Lys Thr 1635 1640 1645 Phe Gly Asn Ser Met Leu Cys Leu Phe GlnIle Thr Thr Ser Ala Gly 1650 1655 1660 Trp Asp Gly Leu Leu Ser Pro IleLeu Asn Thr Gly Pro Pro Tyr Cys 1665 1670 1675 1680 Asp Pro Asn Leu ProAsn Ser Asn Gly Ser Arg Gly Asn Cys Gly Ser 1685 1690 1695 Pro Ala ValGly Ile Ile Phe Phe Thr Thr Tyr Ile Ile Ile Ser Phe 1700 1705 1710 LeuIle Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn Phe Asn 1715 17201725 Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp Phe Asp1730 1735 1740 Met Phe Tyr Glu Thr Trp Glu Lys Phe Asp Pro Glu Ala ThrGln Phe 1745 1750 1755 1760 Ile Ala Phe Ser Ala Leu Ser Asp Phe Ala AspThr Leu Ser Gly Pro 1765 1770 1775 Leu Arg Ile Pro Lys Pro Asn Gln AsnIle Leu Ile Gln Met Asp Leu 1780 1785 1790 Pro Leu Val Pro Gly Asp LysIle His Cys Leu Asp Ile Leu Phe Ala 1795 1800 1805 Phe Thr Lys Asn ValLeu Gly Glu Ser Gly Glu Leu Asp Ser Leu Lys 1810 1815 1820 Thr Asn MetGlu Glu Lys Phe Met Ala Thr Asn Leu Ser Lys Ala Ser 1825 1830 1835 1840Tyr Glu Pro Ile Ala Thr Thr Leu Arg Trp Lys Gln Glu Asp Leu Ser 18451850 1855 Ala Thr Val Ile Gln Lys Ala Tyr Arg Ser Tyr Met Leu His ArgSer 1860 1865 1870 Leu Thr Leu Ser Asn Thr Leu His Val Pro Arg Ala GluGlu Asp Gly 1875 1880 1885 Val Ser Leu Pro Gly Glu Gly Tyr Ser Thr PheMet Ala Asn Ser Gly 1890 1895 1900 Leu Pro Asp Lys Ser Glu Thr Ala SerAla Thr Ser Phe Pro Pro Ser 1905 1910 1915 1920 Tyr Asp Ser Val Thr ArgGly Leu Ser Asp Arg Ala Asn Ile Asn Pro 1925 1930 1935 Ser Ser Ser MetGln Asn Glu Asp Glu Val Ala Ala Lys Glu Gly Asn 1940 1945 1950 Ser ProGly Pro Gln 1955 21 base pairs nucleic acid single linear cDNA 9CAGCTTCGCT CAGAAGTATC T 21 22 base pairs nucleic acid single linear cDNA10 TTCTCGCCGT TCCACACGGA GA 22 4 amino acids amino acid linear peptide11 Phe Arg Leu Met 1 9 amino acids amino acid linear peptide 12 Thr GlnAsp Phe Trp Glu Asn Leu Tyr 1 5 9 amino acids amino acid linear peptide13 Thr Gln Asp Tyr Trp Glu Asn Leu Tyr 1 5 9 amino acids amino acidlinear peptide 14 Thr Gln Asp Cys Trp Glu Arg Leu Tyr 1 5 9 amino acidsamino acid linear peptide 15 Thr Gln Asp Ser Trp Glu Arg Leu Tyr 1 5 9amino acids amino acid linear peptide 16 Thr Gln Asp Phe Trp Glu Arg LeuTyr 1 5 7 amino acids amino acid linear peptide 17 Thr Gln Asp Ser TrpGlu Arg 1 5 15 amino acids amino acid linear peptide 18 Gly Ser Thr AspAsp Asn Arg Ser Pro Gln Ser Asp Pro Tyr Asn 1 5 10 15 10 amino acidsamino acid linear peptide 19 Ser Pro Lys Glu Asn His Gly Asp Phe Ile 1 510 9 amino acids amino acid linear peptide 20 Pro Asn His Asn Gly SerArg Gly Asn 1 5 15 amino acids amino acid linear peptide 21 Arg Leu LeuArg Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu 1 5 10 15 21 base pairsnucleic acid single linear cDNA 22 GCTTGCTGCG GGTCTTCAAG C 21 14 aminoacids amino acid linear peptide 23 Leu Arg Ala Leu Pro Leu Arg Ala LeuSer Arg Phe Glu Gly 1 5 10 21 base pairs nucleic acid single linear cDNA24 ATCGAGACAG AGCCCGCAGC G 21 44 base pairs nucleic acid single linearcDNA 25 ACGGGTGCCG CAAGGACGGC GTCTCCGTGT GGAACGGCGA GAAG 44 45 basepairs nucleic acid single linear cDNA 26 GGCTATCCTT CCTCTTCCAGCTCTCACCCA GGTATGGAGC CAGGT 45 21 base pairs nucleic acid single linearcDNA 27 TCCCGTACGC TGCAGCTCTT T 21 15 base pairs nucleic acid singlelinear cDNA 28 CCCGGGGAAG GCTAC 15 15 base pairs nucleic acid singlelinear cDNA 29 GTCGACACCA GAAAT 15 30 base pairs nucleic acid singlelinear cDNA 30 GGATCCTCTA GAGTCGACCT GCAGAAGGAA 30 24 base pairs nucleicacid single linear cDNA 31 TGACGCAGGA CTCCTGGGAG CGCC 24

What is claimed is:
 1. A purified nucleic acid sequence encoding asodium channel protein wherein said nucleic acid sequence comprises thecoding portion of the nucleic acid sequence shown in SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5 or SEQ ID NO:7.
 2. The purified nucleic acid sequenceof claim 1 wherein said nucleic acid sequence comprises the codingportion of the nucleic acid sequence shown in SEQ ID NO:1.
 3. A vectorcomprising a nucleic acid sequence of claim 1 or
 2. 4. A host celltransformed or transfected with a nucleic acid sequence of claim 1 or 2.5. A method of producing a mammalian sensory neuron sodium channelprotein, wherein the sodium channel is insensitive to tetrodotoxin,comprising expressing a nucleic acid sequence of claim 1 in a host celltransformed with said nucleic acid sequence.
 6. A nucleic acid sequenceof claim 1 wherein said nucleic acid sequence comprises the codingportion of the nucleic acid sequence shown in SEQ ID NO:3.
 7. A vectorcomprising a nucleic acid sequence of claim
 6. 8. A host celltransformed or transfected with a nucleic acid sequence of claim
 6. 9. Amethod of producing a mammalian sensory neuron sodium channel protein,wherein the sodium channel is insensitive to tetrodotoxin, comprisingexpressing the nucleic acid sequence of claim 6 in a host celltransformed with said nucleic acid sequence.
 10. A nucleic acid sequenceof claim 1 wherein said nucleic acid sequence comprises the codingportion of the nucleic acid sequence shown in SEQ ID NO:5.
 11. A vectorcomprising a nucleic acid sequence of claim
 10. 12. A host celltransformed or transfected with a nucleic acid sequence of claim
 10. 13.A method of producing a mammalian sensory neuron sodium channel protein,wherein the sodium channel is insensitive to tetrodotoxin, comprisingexpressing the nucleic acid sequence of claim 10 in a host celltransformed with said nucleic acid sequence.
 14. A nucleic acid sequenceof claim 1 wherein said nucleic acid sequence comprises the codingportion of the nucleic acid sequence shown in SEQ ID NO:7.
 15. A vectorcomprising a nucleic acid sequence of claim
 14. 16. A host celltransformed or transfected with a nucleic acid sequence of claim
 14. 17.A method of producing a mammalian sensory neuron sodium channel protein,wherein the sodium channel is insensitive to tetrodotoxin, comprisingexpression the nucleic acid sequence of claim 14 in a host celltransformed with said nucleic acid.